Compositions and methods for treating osteolytic disorders comprising mmp-14 binding proteins

ABSTRACT

Provided are methods and compositions for using MMP-14 or MMP-9 binding proteins alone or in combination with other therapeutic agents to treat osteolytic disorders such as osteotropic cancer and osteoporosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.61/008,153, filed on Dec. 17, 2007, U.S. Application Ser. No.61/025,032, filed on Jan. 31, 2008, and U.S. Application Ser. No.61/107,510, filed on Oct. 22, 2008. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

BACKGROUND

Osteoclasts, which mediate bone resorption, are involved in normal andabnormal bone remodeling processes, including osteolytic disorders.Osteoclasts are multinucleated cells differentiating from haemopoieticcells. It is generally accepted that osteoclasts are formed by thefusion of mononuclear precursors derived from haemopoietic stem cells inthe bone marrow, rather than incomplete cell division. Thedifferentiation of osteoclast precursors into mature multinucleatedosteoclasts requires different factors including hormonal and localstimuli and living bone and bone cells have been shown to play acritical role in osteoclast development. Osteoblastic or bone marrowstromal cells are also required for osteoclast differentiation.Osteoclasts are responsible for dissolving both the mineral and organicbone matrix. Osteoclasts represent terminally differentiated cellsexpressing a unique polarized morphology with specialized membrane areasand several membrane and cytoplasmic markers.

Several molecular mechanisms bring about cancer cells to metastasize tobone, and osteotropic cancer cells are believed to acquire bonecell-like properties which improve homing, adhesion, proliferation andsurvival in the bone microenvironment. Signaling pathways involved intumor growth and development of osteolytic lesions include RANK, RANKL,osteoprotegerin (OPG), IGF and the membrane type (MT)-matrixmetalloproteinases (MMPs). The initial phase of bone degradationconsists of removal of the unmineralized type I collagenous layerfollowed by degradation of the mineralized matrix, which also comprisestype I collagen. Tumor expansion in bone requires the removal of thismatrix that is particularly abundant and resistant to degradation. Theassistance of osteoclasts appears to be mandatory because osteoclastsare the primary cells involved in bone matrix solubilization. Thecapacity of osteoclasts to degrade bone resides in their ability tosecrete protons, cathepsin K and MMPs. A generalized increase in MMPslevels within the bone environment when cancer cells are present is due,in part, to production of MMPs by the cancer cells themselves.

Since osteoclasts play a major role in osteolytic bone metastases andother osteolytic diseases, there is a need in the art for new agents andmethods for preventing osteoclast stimulation and function. Severaltherapeutic strategies targeting osteolytic disease are currently beingused or under development, where efforts have mainly focused on thedevelopment of drugs to block bone resorption through inhibiting theformation or activity of osteoclasts. The bisphosphonates (BPs),pyrophosphate analogs that concentrate in bone, are to date the mosteffective inhibitor of bone resorption. BPs are taken up by osteoclasts,inhibiting their activity and causing the cells to undergo apoptosis,thereby inhibiting bone resorption. Advanced cancers are prone tometastasize. Effective treatments for bone metastases are not yetavailable—existing treatments such as bisphosphonates, chemotherapy andradiotherapy improve the quality of life with no life-prolongingbenefits and have significant side effects.

SUMMARY

Disclosed herein are methods for the treatment of osteolytic disorders,in particular osteotropic cancer and osteoporosis. In one aspect, theinvention provides methods for the treatment or prevention of anosteolytic disorder comprising administration of an effective amount ofa MMP-14 or MMP-9 binding protein. In certain embodiments wherein theosteolytic disorder is osteotropic cancer, the methods act specificallyto decrease and/or prevent the occurrence of osteolytic lesions whichcan occur due to metastatic spread to bone of a number of cancersincluding but not limited to breast, lung and prostate, byadministration of a MMP-14 or MMP-9 binding protein. In otherembodiments, the methods act specifically to prevent osteolytic lesionsfrom forming in subjects having bone metastases, by administration of aMMP-14 or MMP-9 binding protein.

In one embodiment, an MMP-14 binding protein is administered incombination with an MMP-9 binding protein. In one embodiment, the MMP-14or MMP-9 binding protein is administered in combination with anadditional cancer therapeutic or treatment, such as, for example,bisphosphonates (e.g., amino and non-amino bisphosphonates),hormone-related compounds (e.g., estrogens and SERMs), RANKLantagonists, α_(γ)β₃ antagonists, Src inhibitors, cathepsin Kinhibitors, calcitonin, chemotherapy and radiotherapy.

In one embodiment, an MMP-14 binding protein is administered incombination with an MMP-9 binding protein and an additional cancertherapeutic or treatment, such as, for example, bisphosphonates (e.g.,amino and non-amino bisphosphonates), hormone-related compounds (e.g.,estrogens and SERMs), RANKL antagonists, α_(γ)β₃ antagonists, Srcinhibitors, cathepsin K inhibitors, calcitonin, chemotherapy andradiotherapy.

In one aspect, the invention provides kits for the treatment of anosteolytic disorder. The kits include a MMP-14 and/or MMP-9 bindingprotein, and instructions for administering the MMP-14 and/or MMP-9binding protein to a subject having an osteolytic disorder. In oneembodiment, the kit further includes instructions for administration ofan additional therapeutic for the treatment of an osteolytic disorder,and may optionally contain the additional therapeutic. In oneembodiment, the instructions provide a dosing regimen, dosing schedule,and/or route of administration of the MMP-14 and/or MMP-9 bindingprotein that differs from the dosing regimen, dosing schedule and/orroute of administration for the inhibitor in the absence of theadditional therapeutic.

In another aspect, provided herein is the use of a MMP-14 and/or MMP-9binding protein for the manufacture of a medicament for the treatment ofan osteolytic disorder. The medicament may optionally include anadditional therapeutic for the treatment of an osteolytic disorder, suchas a bisphosphonate.

The MMP-14 and/or MMP-9 binding protein used in any disclosed method,kit or composition can have one or more of the characteristics describedbelow in the Detailed Description. Preferred compositions, e.g., used inany method or kit described herein, may further comprise one or morepharmaceutically acceptable buffers, carriers, and excipients, which mayprovide a desirable feature to the composition including, but notlimited to, enhanced administration of the composition to a patient,enhanced circulating half-life of the inhibitor, enhanced compatibilityof the composition with patient blood chemistry, enhanced storage of thecomposition, and/or enhanced efficacy of the composition uponadministration to a patient.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts amino acid sequences of Fab heavy chain (HC) and lightchain (LC) variable regions of some exemplary MMP-14 binding proteinswhich may be used in the provided methods and compositions for treatingosteolytic disorders. The standard numbering of the HC V domain isshown. The length of HC CDR3 varies considerably. By convention, thesecond cysteine is numbered 92 and the W of the conserved WG motif ofFR4 is number 103. If there are more than 9 residues between C92 andW103, then residues after 102 are numbered 102a, 102b, etc.=

FIG. 2 depicts (left) X-ray analysis of the size of osteolytic tibiallesions treated with a PBS control (top) or DX-2400 (bottom) and (right)bone histomorphometric analysis of the size of osteolytic tibial lesionstreated with a PBS control or DX-2400, as indicated.

FIG. 3 depicts amino acid sequences of Fab heavy chain (HC) and lightchain (LC) variable regions of some exemplary MMP-9 binding proteinswhich may be used in the provided methods and compositions for treatingosteolytic disorders.

DETAILED DESCRIPTION

Expression of MMP-2, MMP-9 and MMP-14 in sections from core bone biopsyspecimens from patients with bone-metatstatic prostate cancer has beenobserved. Further, expression of RANKL, MMP-2, MMP-13 and MMP-14 hasbeen observed to be markedly elevated in bone with metastasis of breastcancer MDA-MB-231 cells in vivo. The disclosure provides methods ofusing MMP-14 or MMP-9 binding proteins, including MMP-14 or MMP-9binding proteins that inhibit MMP-14 or MMP-9 binding activity, in thetreatment and prevention of osteolytic disorders such as osteotropiccancer and osteoporosis, as well as compositions and kits for the same.

DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “antibody” refers to a protein that includes at least oneimmunoglobulin variable domain or immunoglobulin variable domainsequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)₂, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments (de Wildt et al., Eur J. Immunol. 1996;26(3):629-39.)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). Antibodies may be from any source, but primate (human andnon-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk).Kabat definitions are used herein. Each VH and VL is typically composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. In IgGs, the heavy chainconstant region includes three immunoglobulin domains, CH1, CH2 and CH3.The light chain constant region includes a CL domain. The variableregion of the heavy and light chains contains a binding domain thatinteracts with an antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.The light chains of the immunoglobulin may be of types kappa or lambda.In one embodiment, the antibody is glycosylated. An antibody can befunctional for antibody-dependent cytotoxicity and/orcomplement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each ofthe light chain CDRs can be human. HC CDR3 can be human. One or more ofthe framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of theHC or LC. For example, the Fc region can be human. In one embodiment,all the framework regions are human, e.g., derived from a human somaticcell, e.g., a hematopoietic cell that produces immunoglobulins or anon-hematopoietic cell. In one embodiment, the human sequences aregermline sequences, e.g., encoded by a germline nucleic acid. In oneembodiment, the framework (FR) residues of a selected Fab can beconverted to the amino-acid type of the corresponding residue in themost similar primate germline gene, especially the human germline gene.One or more of the constant regions can be human or effectively human.For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of animmunoglobulin variable domain, the constant region, the constantdomains (CH1, CH2, CH3, CL1), or the entire antibody can be human oreffectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the many immunoglobulinvariable region genes. Full-length immunoglobulin “light chains” (about25 KDa or about 214 amino acids) are encoded by a variable region geneat the NH₂-terminus (about 110 amino acids) and a kappa or lambdaconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 KDa or about 446 amino acids), are similarlyencoded by a variable region gene (about 116 amino acids) and one of theother aforementioned constant region genes, e.g., gamma (encoding about330 amino acids). The length of human HC varies considerably because HCCDR3 varies from about 3 amino-acid residues to over 35 amino-acidresidues.

The term “antigen-binding fragment” of a full length antibody refers toone or more fragments of a full-length antibody that retain the abilityto specifically bind to a target of interest. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of afull length antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, abivalent fragment including two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andCH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains functionality.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos.5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883.

Antibody fragments can be obtained using any appropriate techniqueincluding conventional techniques known to those with skill in the art.The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or fragments thereof of single molecular composition,irrespective of how the antibody was generated.

As used herein, “binding affinity” refers to the apparent associationconstant or K_(a). The K_(a) is the reciprocal of the dissociationconstant (K_(s)). A binding protein may, for example, have a bindingaffinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M⁻¹ for aparticular target molecule, e.g., MMP-14, MMP-16, or MMP-24. Higheraffinity binding of a binding protein to a first target relative to asecond target can be indicated by a higher K_(a) (or a smaller numericalvalue K_(d)) for binding the first target than the K_(a) (or numericalvalue K_(d)) for binding the second target. In such cases, the bindingprotein has specificity for the first target (e.g., a protein in a firstconformation or mimic thereof) relative to the second target (e.g., thesame protein in a second conformation or mimic thereof; or a secondprotein). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5,50, 70, 80, 91, 100, 500, 1000, or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inTRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂ at pH7.5). Thesetechniques can be used to measure the concentration of bound and freebinding protein as a function of binding protein (or target)concentration. The concentration of bound binding protein ([Bound]) isrelated to the concentration of free binding protein ([Free]) and theconcentration of binding sites for the binding protein on the targetwhere (N) is the number of binding sites per target molecule by thefollowing equation:

[Bound]=N·[Free]/((1/Ka)+[Free]).

It is not always necessary to make an exact determination of K_(a),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, that is proportional to K_(a), and thus can be usedfor comparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

The term “binding protein” refers to a protein or polypeptide that caninteract with a target molecule. This term is used interchangeably with“ligand.” An “MMP-14 binding protein” refers to a protein that caninteract with MMP-14, and includes, in particular, proteins thatpreferentially interact with and/or inhibit MMP-14. For example, theMMP-14 binding protein may be an antibody. An “MMP-9 binding protein”refers to a protein that can interact with MMP-9, and includes, inparticular, proteins that preferentially interact with and/or inhibitMMP-9. For example, the MMP-9 binding protein may be an antibody.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

The term “cognate ligand” refers to a naturally occurring ligand of anMMP-14 or MMP-9, including naturally occurring variants thereof (e.g.,splice variants, naturally occurring mutants, and isoforms).

The term “combination” refers to the use of the two or more agents ortherapies to treat the same patient, wherein the use or action of theagents or therapies overlap in time. The agents or therapies can beadministered at the same time (e.g., as a single formulation that isadministered to a patient or as two separate formulations administeredconcurrently) or sequentially in any order.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possiblefor many framework and CDR amino acid residues to include one or moreconservative substitutions.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

An “epitope” refers to the site on a target compound that is bound by abinding protein (e.g., an antibody such as a Fab or full lengthantibody). In the case where the target compound is a protein, the sitecan be entirely composed of amino acid components, entirely composed ofchemical modifications of amino acids of the protein (e.g., glycosylmoieties), or composed of combinations thereof. Overlapping epitopesinclude at least one common amino acid residue, glycosyl group,phosphate group, sulfate group, or other molecular feature.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% ofthe length of the reference sequence. For example, the referencesequence may be the length of the immunoglobulin variable domainsequence.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified to include a sufficient number of humanframework amino acid positions such that the immunoglobulin variableregion does not elicit an immunogenic response in a normal human.Descriptions of “humanized” immunoglobulins include, for example, U.S.Pat. No. 6,407,213 and U.S. Pat. No. 5,693,762.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either can beused. Specific hybridization conditions referred to herein are asfollows: (1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); (2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) highstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions and the onesthat should be used unless otherwise specified. The disclosure includesnucleic acids that hybridize with low, medium, high, or very highstringency to a nucleic acid described herein or to a complementthereof, e.g., nucleic acids encoding a binding protein describedherein. The nucleic acids can be the same length or within 30, 20, or10% of the length of the reference nucleic acid. The nucleic acid cancorrespond to a region encoding an immunoglobulin variable domainsequence described herein.

An MMP-14 or MMP-9 binding protein may have mutations (e.g., at leastone, two, or four, and/or less than 15, 10, 5, or 3) relative to abinding protein described herein (e.g., a conservative or non-essentialamino acid substitutions), which do not have a substantial effect onprotein function. Whether or not a particular substitution will betolerated, i.e., will not adversely affect biological properties, suchas binding activity can be predicted, e.g., by evaluating whether themutation is conservative or by the method of Bowie; et al. (1990)Science 247:1306-1310.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain such that one or more CDR regions are positioned in aconformation suitable for an antigen binding site. For example, thesequence may include all or part of the amino acid sequence of anaturally-occurring variable domain. For example, the sequence may omitone, two or more N- or C-terminal amino acids, internal amino acids, mayinclude one or more'insertions or additional terminal amino acids, ormay include other alterations. In one embodiment, a polypeptide thatincludes immunoglobulin variable domain sequence can associate withanother immunoglobulin variable domain sequence to form an antigenbinding site, e.g., a structure that preferentially interacts with anMMP-14 or MMP-9 protein, e.g., the MMP-14 or MMP-9 catalytic domain.

An “isolated composition” refers to a composition that is removed fromat least 90% of at least one component of a natural sample from whichthe isolated composition can be obtained. Compositions producedartificially or naturally can be “compositions of at least” a certaindegree of purity if the species or population of species of interests isat least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on aweight-weight basis.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas changing an “essential” amino acid residueresults in a substantial loss of activity.

As used herein, the phrase “metastatic cancer” is defined as a cancerthat has potential to spread to other areas of the body, particularlybone. A variety of cancers can metastasize to the bone, but the mostcommon metastasizing cancers are breast, lung, renal, multiple myeloma,thyroid and prostate. By way of example, other cancers that have thepotential to metastasize to bone include but are not limited toadenocarcinoma, blood cell malignancies, including leukemia andlymphoma; head and neck cancers; gastrointestinal cancers, includingesophageal cancer, stomach cancer, colon cancer, intestinal cancer,colorectal cancer, rectal cancer, pancreatic cancer, liver cancer,cancer of the bile duct or gall bladder; malignancies of the femalegenital tract, including ovarian carcinoma, uterine endometrial cancers,vaginal cancer, and cervical cancer; bladder cancer; brain cancer,including neuroblastoma; sarcoma, osteosarcoma; and skin cancer,including malignant melanoma and squamous cell cancer. The presentinvention especially contemplates prevention and treatment oftumor-induced osteolytic lesions in bone.

As used herein, an “osteolytic disorder” is any condition resulting fromincreased osteoclast activity. A subject at risk of an osteolyticdisorder may be a subject in a group predisposed to develop anosteolytic disorder, or a subject suffering from a disease that causesor contributes to increased osteoclastic activity. In exemplaryembodiments of the invention, the osteolytic disorder maybe a metabolicbone disease associated with relatively increased osteoclast activity,including an endocrinopathy (hypercortisolism, hypogonadism, primary orsecondary hyperparathyroidism, hyperthyroidism), hypercalcemia,deficiency state (rickets/osteomalacia, scurvy, malnutrition), chronicdisease (malabsorption syndromes, chronic renal failure (renalosteodystrophy), chronic liver disease (hepatic osteodystrophy)), drugs(glucocorticoids (glucocorticoid-induced osteoporosis), heparin,alcohol), or hereditary disease (osteogenesis imperfecta,homocystinuria), cancer, osteoporosis, osteopetrosis, inflammation ofbone associated with arthritis and rheumatoid arthritis, periodontaldisease, fibrous dysplasia, and/or Paget's disease. In other exemplaryembodiments, the osteolytic disorder may be a metastatic cancer to bone(osteotropic cancer), wherein the metastatic cancer is breast, lung,renal, multiple myeloma, thyroid, prostate, adenocarcinoma, blood cellmalignancy, including leukemia and lymphoma; head and neck cancer;gastrointestinal cancer, including esophageal cancer, stomach cancer,colon cancer, intestinal cancer, colorectal cancer, rectal cancer,pancreatic cancer, liver cancer, cancer of the bile duct or gallbladder; malignancy of the female genital tract, including ovariancarcinoma, uterine endometrial cancer, vaginal cancer, or cervicalcancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma,osteosarcoma; or skin cancer, including malignant melanoma or squamouscell cancer. In some embodiments, increased osteoclast activity, e.g.,in a subject, refers to osteoclast activity that is increased ascompared to the levels of osteoclast activity in a standard, e.g., theosteoclast activity in a cohort of subjects, e.g., a cohort of subjectswithout a symptom of an osteoclast disorder, or the levels of osteoclastactivity of a random sampling of subjects. Osteoclast activity canincrease by, e.g., about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or more, as compared tothe standard. Osteoclast activity can be measured, e.g., by tartrateresistant acid phosphatase (TRAP) staining; ELISA analysis of ReceptorActivator for Nuclear Factor κB Ligand (RANKL) concentration in bloodserum; and/or Alizarin Red staining of osteoblastic cells which wereisolated from bone marrow. Additional methods are described, e.g., in WO2003/031597.

The term “osteoporosis” refers to a disease in which the bones become,extremely porous, are subject to fracture, and heal slowly, occurringespecially in women following menopause and often leading to curvatureof the spine from vertebral collapse.

The term “osteotropic cancer” refers to metastatic cancer of the bone,i.e., a secondary cancer present in bone that originates from a primarycancer, such as that of the breast, lung, or prostate.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term “preventing” a disease in a subject refers to subjecting thesubject to a pharmaceutical treatment, e.g., the administration of adrug, such that at least one symptom of the disease is prevented, thatis, administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal) sothat it protects the host against developing the unwanted condition.“Preventing” a disease may also be referred to as “prophylaxis” or“prophylactic treatment.”

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, because a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleicacid sequence that contains a sufficient number of identical orequivalent (e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleic acid sequence such that the first and second amino acid ornucleic acid sequences have (or encode proteins having) similaractivities, e.g., a binding activity, a binding preference, or abiological activity. In the case of antibodies, the second antibody hasthe same specificity and has at least 50%, at least 25%, or at least 10%of the affinity relative to the same antigen. Sequences similar orhomologous (e.g., at least about 85% sequence identity) to the sequencesdisclosed herein are also part of this application. In some embodiments,the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher. In addition, substantial identity existswhen the nucleic acid segments hybridize under selective hybridizationconditions (e.g., highly stringent hybridization conditions), to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form.

Motif sequences for biopolymers can include positions which can bevaried amino acids. For example, the symbol “X” in such a contextgenerally refers to any amino acid (e.g., any of the twenty naturalamino acids or any of the nineteen non-cysteine amino acids). Otherallowed amino acids can also be indicated for example, using parenthesesand slashes. For example, “(A/W/F/N/Q)” means that alanine, tryptophan,phenylalanine, asparagine, and glutamine are allowed at that particularposition.

Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02. Particular binding proteins may show a difference, e.g., inspecificity or binding, that are statistically significant (e.g., Pvalue<0.05 or 0.02).

The terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”,“decrease” or the like, e.g., which denote distinguishable qualitativeor quantitative differences between two states, may refer to adifference, e.g., a statistically significant difference, between thetwo states.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the proteinto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the composition is outweighed by the therapeutically beneficialeffects.

A “therapeutically effective dosage” preferably modulates a measurableparameter, e.g., levels of circulating IgG antibodies by a statisticallysignificant degree or at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to modulate a measurable parameter, e.g., adisease-associated parameter, can be evaluated in an animal model systempredictive of efficacy in human disorders and conditions, e.g., a cancer(e.g., metastatic cancer, e.g., metastatic breast cancer, e.g.,osteotropic cancer), an inflammatory disease (e.g., synovitis,atherosclerosis), rheumatoid arthritis, osteoarthritis, an ocularcondition (e.g., macular degeneration), diabetes, Alzheimer's Disease,cerebral ischemia, endometriosis, fibrin-invasive activity,angiogenesis, or capillary tube formation. Alternatively, this propertyof a composition can be evaluated by examining the ability of thecompound to modulate a parameter in vitro.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that at least one symptom ofthe disease is cured, alleviated or decreased.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

MMP-14 Binding Proteins

MMP-14 expression is observed in bone samples from patients withbone-metastatic prostate cancer. Any MMP-14 binding protein, e.g., anMMP-14 binding protein described herein, may be used in the methods andcompositions for treating osteolytic disorders that are disclosedherein. MMP-14 is encoded by a gene designated as MMP14, matrixmetalloproteinase-14 precursor. Synonyms for MMP-14 include matrixmetalloproteinase 14 (membrane-inserted), membrane-type-1 matrixmetalloproteinase, membrane-type matrix metalloproteinase 1, MMP-14,MMP14, MMP-X1, MT1MMP, MT1-MMP, MTMMP1, MT-MMP 1. MT-MMPs have similarstructures, including a signal peptide, a prodomain, a catalytic domain,a hinge region, and a hemopexin domain (Wang, et al., 2004, J Biol Chem,279:51148-55). According to SwissProt entry P50281, the signal sequenceof MMP-14 precursor includes amino acid residues 1-20. The pro-peptideincludes residues 21-111. Cys93 is annotated as a possible cysteineswitch. Residues 112 through 582 make up the mature, active protein. Thecatalytic domain includes residues 112-317. The hemopexin domainsincludes residues 318-523. The transmembrane segment comprises residues542 through 562.

MMP-14 can be shed from cells or found on the surface of cells, tetheredby a single transmembrane amino-acid sequence. See, e.g., Osnkowski etal. (2004, J Cell Physiol, 200:2-10).

The MMP-14 binding protein may be an isolated protein (e.g., at least70, 80, 90, 95, or 99% free of other proteins).

The MMP-14 binding protein may additionally inhibit MMP-14, e.g., humanMMP-14. In one embodiment, the protein binds the catalytic domain ofhuman MMP-14, e.g., the protein contacts residues in or near the activesite of MMP-14.

In certain embodiments, proteins that bind to MMP-14 (e.g., humanMMP-14) and include at least one immunoglobulin variable region are usedin the methods and compositions. For example, the MMP-14 binding proteinincludes a heavy chain (HC) immunoglobulin variable domain sequence anda light chain (LC) immunoglobulin variable domain sequence. A number ofexemplary MMP-14 binding proteins are described herein.

MMP-14 binding proteins may also be antibodies. MMP-14 bindingantibodies may have their HC and LC variable domain sequences includedin a single polypeptide (e.g., scFv), or on different polypeptides(e.g., IgG or Fab). For example, antibodies may be raised against any ofthe following sequences:

An exemplary amino acid sequence of human MMP-14 is shown in Table 1:

TABLE 1 Amino-acid sequence of human MMP-14(SEQ ID NO: 1; Genbank Accession No. CAA88372.1)MSPAPRPPRCLLLPLLTLGTALASLGSAQSSSFSPEAWLQQYGYLPPGDLRTHTQRSPQSLSAAIAAMQKFYGLQVTGKADADTMKAMRRPRCGVPDKFGAEIKANVRRKRYAIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGFPTKMPPQPRTTSRPSVPDKPKNPTYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQFWRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALFWMPNGKTYFFRGNKYYRFNEELRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKSALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGGGAVSAAAVVLPVLLLLLVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKV.

An exemplary amino acid sequence of mouse MMP-14 is shown in Table 2.

TABLE 2 Amino-acid sequence of mouse MMP-14SEQ ID NO: 2; GenBank Accession No. NP_032634.2MSPAPRPSRSLLLPLLTLGTALASLGWAQGSNFSPEAWLQQYGYLPPGDLRTHTQRSPQSLSAAIAAMQKFYGLQVTGKADLATMMAMRRPRCGVPDKFGTEIKANVRRKRYAIQGLKWQHNEITFCIQNYTPKVGEYATFEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMILFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVQNEDLNGNDIFLVAVHELGHALGLEHSNDPSAIMSPFYQWMDTENFVLPDDDRRGIQQLYGSKSGSPTKMPPQPRTTSRPSVPDKPKNPAYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQFWRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALFWMPNGKTYFFRGNKYYRFNEEFRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKSALRDWMGCPSGRRPDEGTEEETEVIIIEVDEEGSGAVSAAAVVLPVLLLLLVLAVGLAVFFFRRHGTPKRLLYCQRSLLDKV.

An exemplary MMP-14 protein against which MMP-14 binding proteins may bedeveloped can include the human or mouse MMP-14 amino acid sequence, asequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toone of these sequences, or a fragment thereof, e.g., a fragment withoutthe signal sequence or prodomain.

Exemplary MMP-14 binding proteins include M0031-C02, M0031-F01,M0033-H07, M0037-009, M0037-D01, M0038-E06, M0038-F01, M0038-F08,M0039-H08, M0040-A06, M0040-A11, and M0043-G02. The amino acid sequencesof exemplary Fab heavy chain (HC) and light chain (LC) variable regionsof these binding proteins are shown in FIG. 1, and further descriptionof them and their discovery and production is provided in pendingapplication U.S. Ser. No. 11/648,423 (U.S. 2007-0217997), which ishereby incorporated by reference in its entirety. Other exemplary MMP-14binding proteins include DX-2400 and DX-2410. DX-2400 and M0038-F01share HC and LC CDR amino acid sequences. The amino acid sequences ofthe heavy chain and light chain variable regions of these proteins areprovided in the Examples.

Other MMP-14 inhibitors known in the art include, but are not limitedto, those disclosed in the following patents and patent applications:U.S. Pat. No. 6,114,159; U.S. Pat. No. 6,399,348; JP 3802560 and EP0750672 (all in the name of Max Delbrueck Center for MolecularMedicine); U.S. Pat. No. 6,184,022; U.S. Pat. No. 6,825,024; EP 0685557;JP 2694604 (all in the name of Daiichi Fine Chemicals); and U.S.Provisional Application Ser. Nos. 60/755,376 and 60/805,567 (both in thename of Dyax Corp.).

MMP-9 and MMP-9 Binding Entities

Any MMP-9 binding protein may be used in the methods and compositionsfor treating osteolytic disorders that are disclosed herein.

MMP-9 is encoded by a gene designated as MMP9 with full name Matrixmetalloproteinase-9 precursor. Synonyms for MMP-9 include matrixmetalloproteinase 9, gelatinase B (GELB), 92 kDa gelatinase (CLG4B), 92kDa type W collagenase (EC 3.4.24.35). The DNA sequence is known forHomo sapiens and Mus musculus. An exemplary cDNA sequence encoding humanMMP9 and the amino acid sequence are shown below. Exemplary cDNAsequences encoding murine MMP9 and amino acid sequences are also shownbelow. An exemplary MMP-9 protein can include the human or mouse MMP-9amino acid sequence, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to one of these sequences, or a fragment thereof,e.g., a fragment without the signal sequence or prodomain.

Table 3 shows the similar genes in other organisms and the percentage ofsimilarity with human MMP-9. No similarity-to-human data found for MMP9for: chimpanzee (Pan troglodytes), pig (Sus scrofa), cow (Bos taurus),fruit fly (Drosophila melanogaster), worm (Caenorhabditis elegans),baker's yeast (Saccharomyces cerevisiae), tropical clawed frog (Siluranatropicalis), African malaria mosquito (Anopheles gambiae), green algae(Chlamydomonas reinhardtii), soybean (Glycine max), barley (Hordeumvulgare), tomato (Lycopersicon esculentum), rice blast fungus(Magnaporthe grisea), sugarcane (Saccharum officinarum), loblolly pine(Pinus taeda), corn (Zea mays), wheat (Triticum aestivum), Alicantegrape (Vitis vinifera), bread mold (Neurospora crassa), fission yeast(Schizosaccharomyces pombe), sea squirt (Ciona intestinalis), amoeba(Dictyostelium discoideum), A. gosspyii yeast (Ashbya gossypii), K.lactis yeast (Kluyveromyces lactis), medicago trunc (Medicagotruncatula), malaria parasite (Plasmodium falciparum), schistosomeparasite (Schistosoma mansoni), sorghum (Sorghum bicolor), toxoplasmosis(Toxoplasma gondii).

cDNA and amino acid sequences of human MMP9 ACCESSION  AK123156VERSION    AK123156.1  GI:34528630 (SEQ ID NO: 3) translatin =“MARKGARRPRQGPGSHKWLQPGSRREKERIPQPPPPARPPRDAAPRRVLVPAVRRVPESGHFAGRPWAPQCHPKGLRRPSAESHSVAQAGVQCHDLGSLQPPPPSSGDSPASASRVAGITSTVPGTLSALDDCCLITELPYKPPAVLY” (SEQ ID NO: 4) 1acactttgcg ttccgcggcc ccggcccctt ggtttcctag tcctggctcc attcccctct 61caggcctagg gctgggaccc ctccccgccc ccggtcttgg ccctgccccc ttcaacagac 121ggtccgcccc ggcccctccc cctcgtcccg cccggccctg gcaggccccg ccccctgcgg 181cctctacctt tgacgtcttc ccccgggagg tggcgggggt ctgcgaccga atgccggcgg 241gactctgggt cagggcttct ggcgggccct gcggggggca gcgaggtgac cgtgaacctg 301cggctcatgg cgcggaaagg agccaggcgg ccgcggcaag gtccgggatc gcacaagtgg 361ctgcaaccag gctctaggag ggagaaagag cggatccccc aaccccctcc gcccgcccgc 421cccccgcgag acgcggcgcc gcgcagggtc ctagtgcccg ctgtgcgaag ggttcctgaa 481tctggccact tcgctgggag gccctgggct ccccagtgcc acccgaaggg cctgaggagg 541ccatctgcag aatctcactc tgtcgcccag gccggagtgc agtgtcatga tcttggctca 601ctgcaacctc cgcctcccag ttcaggagat tctcctgcct cagcctcccg ggtggctggg 661attacaagca cagtgcctgg cacattatcg gcacttgatg actgttgtct aataactgag 721cttccataca aaccacctgc cgtcctgtac tgaaggagaa agagcttcca gccggggagg 781caggaaatct gggtcctggt cttggttgca tccctgactt cctaaatgac ctggagaagg 841cctctgcctc tgctgggatc ttgtctgtgc tggggcattt gtttccattt ccaagggctt 901tttcttcctc gctcagaatt tgaccactca ctaagaggag cttagtgtgg tgtctcacga 961agggatcctc ctcagccctc acctcggtac tggaagacgt cgtgcgtgtc caaaggcacc 1021ccggggaaca tccggtccac ctcgctggcg ctccggggat ccaccatctg cgccttcacg 1081tcgaacctgc gggcaggcgc ggaggagaca ggtgctgagc cggctagcgg acggaccgac 1141ggcgcccggg ctccccctgc cggcggccgc ggcggcgctc acctccagag gcgccgcccg 1201ctgaacagca gcatcttccc cctgccactc cggagggccc cggtcacctg ggccacgtcg 1261gcgcccaggc ccagcttgtc cagacgcctc gggcccagca ccgacgcgcc tgtgtacacc 1321cacacctggc gccctgcagg ggaggagggt cacgtcggtt tgggggcgca gagggagcac 1381gtactcctag aacgcgagga gggagattcc ggcgaggcct ttcctagccc gcgtgcccgc 1441agtccctgca acccaggggc agaggcgctg ggtagagcga cgcgagggcg tggagaggag 1501ggggcagaaa ctcagccgcc cctacgtttg ctaaactgcg tccgccaggg ggcgtatttt 1561tctaaaacgc acaagacgtt tcgtgggtta tcgatggtct cttgagcctc cttgactgat 1621ggggattgac cgggcggggg agggaaagta ggtaactaac cagagaagaa gaaaagcttc 1681ttggagagcg gctcctcaaa gaccgagtcc agcttgcggg gcagcgcggg ccacttgtcg 1741gcgataagga aggggccctg cggccggctc cccctgccct cagagaatcg ccagtacttc 1801ctgagaaagc gaggagggaa aggacgggct ctaagccttg gacacagggc cagtgggcgg 1861gaagggacgg gcagcccctc cgcaaagccc cctcccgcat ccacacaacc ccgcctcctc 1921acccatcctt gaacaaatac agctggttcc caatccDNA and amino acid sequences of mouse MMP9 ACCESSION  NM_013599VERSION    NM_013599.2  GI:31560795 (SEQ ID NO: 5) translation =“MSPWQPLLLALLAFGCSSAAPYQRQPTFVVFPKDLKTSNLTDTQLAEAYLYRYGYTRAAQMMGEKQSLRPALLMLQKQLSLPQTGELDSQTLKAIRTPRCGVPDVGRFQTFKGLKWDHHNITYWIQNYSEDLPRDMIDDAFARAFAVWGEVAPLTFTRVYGPEADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGAGVQGDAHFDDDELWSLGKGVVIPTYYGNSNGAPCHFPFTFEGRSYSACTTDGRNDGTPWCSTTADYDKDGKFGFCPSERLYTEHGNGEGKPCVFPFIFEGRSYSACTTKGRSDGYRWCATTANYDQDKLYGFCPTRVDATVVGGNSAGELCVFPFVFLGKQYSSCTSDGRRDGRLWCATTSNFDTDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPLYSYLEGFPLNKDDIDGIQYLYGRGSKPDPRPPATTTTEPQPTAPPTMCPTIPPTAYPTVGPTVGPTGAPSPGPTSSPSPGPTGAPSPGPTAPPTAGSSEASTESLSPADNPCNVDVFDAIAEIQGALHFFKDGWYWKFLNHRGSPLQGPFLTARTWPALPATLDSAFEDPQTKRVFFFSGRQMWVYTGKTVLGPRSLDKLGLGPEVTHVSGLLPRRLGKALLFSKGRVWRFDLKSQKVDPQSVIRVDKEFSGVPWNSHDIFQYQDKAYFCHGKFFWRVSFQNEVNKVDHEVNQVDDVGYVTYDLLQCP” (SEQ ID NO: 6) 1ctcaccatga gtccctggca gcccctgctc ctggctctcc tggctttcgg ctgcagctct 61gctgcccctt accagcgcca gccgactttt gtggtcttcc ccaaagacct gaaaacctcc 121aacctcacgg acacccagct ggcagaggca tacttgtacc gctatggtta cacccgggcc 181gcccagatga tgggagagaa gcagtctcta cggccggctt tgctgatgct tcagaagcag 241ctctccctgc cccagactgg tgagctggac agccagacac taaaggccat tcgaacacca 301cgctgtggtg tcccagacgt gggtcgattc caaaccttca aaggcctcaa gtgggaccat 361cataacatca catactggat ccaaaactac tctgaagact tgccgcgaga catgatcgat 421gacgccttcg cgcgcgcctt cgcggtgtgg ggcgaggtgg cacccctcac cttcacccgc 481gtgtacggac ccgaagcgga cattgtcatc cagtttggtg tcgcggagca cggagacggg 541tatcccttcg acggcaagga cggccttctg gcacacgcct ttccccctgg cgccggcgtt 601cagggagatg cccatttcga cgacgacgag ttgtggtcgc tgggcaaagg cgtcgtgatc 661cccacttact atggaaactc aaatggtgcc ccatgtcact ttcccttcac cttcgaggga 721cgctcctatt cggcctgcac cacagacggc cgcaacgacg gcacgccttg gtgtagcaca 781acagctgact acgataagga cggcaaattt ggtttctgcc ctagtgagag actctacacg 841gagcacggca acggagaagg caaaccctgt gtgttcccgt tcatctttga gggccgctcc 901tactctgcct gcaccactaa aggccgctcg gatggttacc gctggtgcgc caccacagcc 961aactatgacc aggataaact gtatggcttc tgccctaccc gagtggacgc gaccgtagtt 1021gggggcaact cggcaggaga gctgtgcgtc ttccccttcg tcttcctggg caagcagtac 1081tcttcctgta ccagcgacgg ccgcagggat gggcgcctct ggtgtgcgac cacatcgaac 1141ttcgacactg acaagaagtg gggtttctgt ccagaccaag ggtacagcct gttcctggtg 1201gcagcgcacg agttcggcca tgcactgggc ttagatcatt ccagcgtgcc ggaagcgctc 1261atgtacccgc tgtatagcta cctcgagggc ttccctctga ataaagacga catagacggc 1321atccagtatc tgtatggtcg tggctctaag cctgacccaa ggcctccagc caccaccaca 1381actgaaccac agccgacagc acctcccact atgtgtccca ctatacctcc cacggcctat 1441cccacagtgg gccccacggt tggccctaca ggcgccccct cacctggccc cacaagcagc 1501ccgtcacctg gccctacagg cgccccctca cctggcccta cagcgccccc tactgcgggc 1561tcttctgagg cctctacaga gtctttgagt ccggcagaca atccttgcaa tgtggatgtt 1621tttgatgcta ttgctgagat ccagggcgct ctgcatttct tcaaggacgg ttggtactgg 1681aagttcctga atcatagagg aagcccatta cagggcccct tccttactgc ccgcacgtgg 1741ccagccctgc ctgcaacgct ggactccgcc tttgaggatc cgcagaccaa gagggttttc 1801ttcttctctg gacgtcaaat gtgggtgtac acaggcaaga ccgtgctggg ccccaggagt 1861ctggataagt tgggtctagg cccagaggta acccacgtca gcgggcttct cccgcgtcgt 1921ctcgggaagg ctctgctgtt cagcaagggg cgtgtctgga gattcgactt gaagtctcag 1981aaggtggatc cccagagcgt cattcgcgtg gataaggagt tctctggtgt gccctggaac 2041tcacacgaca tcttccagta ccaagacaaa gcctatttct gccatggcaa attcttctgg 2101cgtgtgagtt tccaaaatga ggtgaacaag gtggaccatg aggtgaacca ggtggacgac 2161gtgggctacg tgacctacga cctcctgcag tgcccttgaa ctagggctcc ttctttgctt 2221caaccgtgca gtgcaagtct ctagagacca ccaccaccac caccacacac aaaccccatc 2281cgagggaaag gtgctagctg gccaggtaca gactggtgat ctcttctaga gactgggaag 2341gagtggaggc aggcagggct ctctctgccc accgtccttt cttgttggac tgtttctaat 2401aaacacggat ccccaacctt ttccagctac tttagtcaat cagcttatct gtagttgcag 2461atgcatccga gcaagaagac aactttgtag ggtggattct gaccttttat ttttgtgtgg 2521cgtctgagaa ttgaatcagc tggcttttgt gacaggcact tcaccggcta aaccacctct 2581cccgactcca gcccttttat ttattatgta tgaggttatg ttcacatgca tgtatttaac 2641ccacagaatg cttactgtgt gtcgggcgcg gctccaaccg ctgcataaat attaaggtat 2701tcagttgccc ctactggaag gtattatgta actatttctc tcttacattg gagaacacca 2761ccgagctatc cactcatcaa acatttattg agagcatccc tagggagcca ggctctctac 2821tgggcgttag ggacagaaat gttggttctt ccttcaagga ttgctcagag attctccgtg 2881tcctgtaaat ctgctgaaac cagaccccag actcctctct ctcccgagag tccaactcac 2941tcactgtggt tgctggcagc tgcagcatgc gtatacagca tgtgtgctag agaggtagag 3001ggggtctgtg cgttatggtt caggtcagac tgtgtcctcc aggtgagatg acccctcagc 3061tggaactgat ccaggaagga taaccaagtg tcttcctggc agtctttttt aaataaatga 3121ataaatgaat atttacttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3181aaaaa ACCESSION  NP_038627 VERSION    NP_038627.1  GI:7305277(SEQ ID NO: 7) 1mspwqpllla llafgcssaa pygrqptfvv fpkdlktsnl tdtqlaeayl yrygytraaq 61mmgekqslrp allmlqkqls lpqtgeldsq tlkairtprc gvpdvgrfqt fkglkwdhhn 121itywignyse dlprdmidda farafavwge vapltftrvy gpeadiviqf gvaehgdgyp 181fdgkdgllah afppgagvqg dahfdddelw slgkgvvipt yygnsngapc hfpftfegrs 241ysacttdgrn dgtpwcstta dydkdgkfgf cpserlyteh gngegkpcvf pfifegrsys 301acttkgrsdg yrwcattany dqdklygfcp trvdatvvgg nsagelcvfp fvflgkqyss 361ctsdgrrdgr lwcattsnfd tdkkwgfcpd qgyslflvaa hefghalgld hssvpealmy 421plysylegfp lnkddidgiq ylygrgskpd prppatttte pqptapptmc ptipptaypt 481vgptvgptga pspgptssps pgptgapspg ptapptagss easteslspa dnpcnvdvfd 541aiaeiqgalh ffkdgwywkf lnhrgsplqg pfltartwpa lpatldsafe dpqtkrvfff 601sgrqmwvytg ktvlgprsld klglgpevth vsgllprrlg kallfskgry wrfdlksqkv 661dpqsvirvdk efsgvpwnsh difqyqdkay fchgkffwrv sfqnevnkvd hevnqvddvg 721yvtydllqcp

TABLE 3 MMP-9 orthologs from nine species Human Organism Gene LocusDescription Similarity s dog MMP9¹ — matrix metallopeptidase 9 85.46(n)403885 NM 001003219.1 (Canis (gelatinase B, 92 kDa 80.97(a) NP001003219.1 familiaris) gelatinase rat (Rattus Mmp9¹ — matrixmetallopeptidase 9 79.15(n) 81687 NM 031055.1 norvegicus) 74.89(a) NP112317.1 mouse Mmp9^(1, 4) 2 (96.00 cM)⁴ matrix metallopeptidase9^(1, 4)  78.69(n)¹ 17395¹ NM 013599.2¹ (Mus     75(a)¹ NP 038627.1¹musculus) AK004651⁴ AK142787⁴ (see all 16) chicken LOC395387¹ — matrixmetallopeptidase 9 66.96(n) 395387 NM 204667.1 (Gallus (gelatinase B, 92kDa 62.54(a) NP 989998.1 gallus) gelatinase zebrafish wufb02g06^(1~) —Danio rerio cDNA clone 70.96(n) BC053292.1 (Danio rerio) MGC64165IMAGE6797338, complete African MGC69080^(1~) — hypothetical protein72.25(n) BC057745.1 clawed frog MGC69080 (Xenopus laevis) rainbowOmy.10476^(1~) — Oncorhynchus mykiss 74.67(n) AJ320533.1 trout mRNA formatrix (Oncorhynchus metalloproteinase mykiss) thale cress MMP¹ — MMP(MATRIX   53(n) 843353 NM 105685.3 (Arabidopsis METALLOPROTEINASE);46.85(a) NP 177174.1 thaliana) metalloendopeptidase/ rice (OryzaP0516G10.18¹ — putative zinc 51.98(n) 3063368 XM 467714.1 sativa)metalloproteinase 41.81(a) XP 467714.1

Domains of MMP-9. MMP-9 belongs to the peptidase M10A family. MMP-9consists of five domains; the amino-terminal and zinc-binding domainsshared by all members of the secreted metalloprotease gene family, thecollagen-binding fibronectin-like domain also present in the 72-kDa typeIV collagenase, a carboxyl-terminal hemopexin-like domain shared by allknown enzymes of this family with the exception of PUMP-1, and a unique54-amino-acid-long proline-rich domain homologous to the alpha 2 chainof type V collagen (Wilhelm et al. (1989) J. Biol. Chem. 264,17213-17221) (Table 4).

TABLE 4 MMP-9 domains FT SIGNAL 1 19 FT PROPEP 20 93 Activation peptide.FT CHAIN 94 ? 67 kDa matrix metalloproteinase-9. FT CHAIN 107 707 82 kDamatrix metalloproteinase-9. FT PROPEP ? 707 Removed in 64 kDa matrix FTmetalloproteinase-9 and 67 kDa matrix FT metalloproteinase-9. FT DOMAIN225 273 Fibronectin type-II 1. FT DOMAIN 283 331 Fibronectin type-II 2.FT DOMAIN 342 390 Fibronectin type-II 3. FT DOMAIN 513 707Hemopexin-like. FT ACT_SITE 402 402 FT METAL 131 131 Calcium 1. FT METAL165 165 Calcium 2 (via carbonyl oxygen). FT METAL 175 175 Zinc 1(structural). FT METAL 177 177 Zinc 1 (structural). FT METAL 182 182Calcium 3. FT METAL 183 183 Calcium 3 (via carbonyl oxygen). FT METAL185 185 Calcium 3 (via carbonyl oxygen). FT METAL 187 187 Calcium 3 (viacarbonyl oxygen). FT METAL 190 190 Zinc 1 (structural). FT METAL 197 197Calcium 2 (via carbonyl oxygen). FT METAL 199 199 Calcium 2 (viacarbonyl oxygen). FT METAL 201 201 Calcium 2. FT METAL 203 203 Zinc 1(structural). FT METAL 205 205 Calcium 3. FT METAL 206 206 Calcium 1. FTMETAL 208 208 Calcium 1. FT METAL 208 208 Calcium 3. FT METAL 401 401Zinc 2 (catalytic). FT METAL 405 405 Zinc 2 (catalytic). FT METAL 411411 Zinc 2 (catalytic). FT SITE 59 60 Cleavage (by MMP3). FT SITE 99 99Cysteine switch (By similarity). FT SITE 106 107 Cleavage (by MMP3). FTCARBOHYD 38 38 N-linked (GlcNAc . . .) (Potential). FT CARBOHYD 120 120N-linked (GlcNAc . . .) (Potential). FT CARBOHYD 127 127 N-linked(GlcNAc . . .) (Potential). FT DISULFID 230 256 By similarity. FTDISULFID 244 271 By similarity. FT DISULFID 288 314 By similarity. FTDISULFID 302 329 By similarity. FT DISULFID 347 373 By similarity. FTDISULFID 361 388 By similarity. FT DISULFID 516 704 FT VARIANT 20 20 A−> V (in dbSNP: rs1805088). FT VARIANT 82 82 E −> K (in dbSNP:rs1805089). FT VARIANT 127 127 N −> K (in dbSNP: rs3918252). FT VARIANT239 239 R −> H. FT VARIANT 279 279 R −> Q (common polymorphism; FTdbSNP: rs17576). FT VARIANT 571 571 F −> V. FT VARIANT 574 574 P −> R(in dbSNP: rs2250889). FT VARIANT 668 668 R −> Q (in dbSNP: rs17577). FTTURN 32 33 FT HELIX 41 51 FT TURN 52 53 FT HELIX 68 78 FT TURN 79 79 FTHELIX 88 94 FT TURN 95 95 FT STRAND 103 105 FT STRAND 119 125 FT STRAND130 132 FT HELIX 134 149 FT TURN 150 150 FT STRAND 151 153 FT STRAND 155158 FT TURN 162 163 FT STRAND 164 171 FT STRAND 176 178 FT STRAND 183186 FT STRAND 189 191 FT STRAND 194 196 FT TURN 197 200 FT STRAND 202205 FT TURN 206 207 FT STRAND 213 219 FT HELIX 220 231 FT TURN 232 233FT TURN 240 241 FT TURN 243 244 FT STRAND 245 247 FT STRAND 255 261 FTHELIX 262 265 FT STRAND 268 270 FT TURN 274 276 FT STRAND 279 283 FTTURN 284 285 FT STRAND 290 294 FT TURN 295 296 FT STRAND 297 301 FT TURN305 306 FT STRAND 313 319 FT HELIX 320 323 FT STRAND 326 328 FT HELIX333 335 FT TURN 340 344 FT STRAND 349 353 FT TURN 354 355 FT STRAND 356358 FT TURN 364 365 FT STRAND 372 378 FT HELIX 379 382 FT STRAND 385 387FT HELIX 395 406 FT TURN 407 408 FT TURN 415 416 FT TURN 418 419 FTHELIX 433 442 FT STRAND 512 517 FT HELIX 515 517 FT STRAND 522 527 FTTURN 528 529 FT STRAND 530 535 FT TURN 536 537 FT STRAND 538 542 FTSTRAND 545 547 FT STRAND 551 555 FT HELIX 556 559 FT TURN 561 562 FTSTRAND 568 572 FT TURN 574 576 FT STRAND 579 583 FT TURN 584 585 FTSTRAND 586 591 FT TURN 592 593 FT STRAND 594 600 FT HELIX 601 604 FTTURN 605 605 FT TURN 608 609 FT STRAND 615 618 FT TURN 621 622 FT STRAND623 628 FT TURN 629 630 FT STRAND 631 636 FT TURN 637 640 FT HELIX 644646 FT HELIX 650 653 FT TURN 655 656 FT STRAND 662 667 FT TURN 668 669FT STRAND 670 675 FT TURN 676 677 FT STRAND 678 683 FT TURN 686 687 FTSTRAND 690 696 FT TURN 697 700 FT TURN 702 703

The catalytic activity of MMP-9 is inhibited by histatin-3 1/24(histatin-5). MMP-9 is activated by urokinase-type plasminogenactivator; plasminogen; IL-1beta, 4-aminophenylmercuric acetate andphorbol ester. MMP-9 exists as monomer, disulfide-linked homodimer, andas a heterodimer with a 25 kDa protein. Macrophages and transformed celllines produce only the monomeric MMP-9, the hetrodimeric form isproduced by normal alveolar macrophages and granulocytes. The processingof the precursor yields different active forms of 64, 67 and 82 kDa.Sequentially processing by MMP-3 yields the 82 kDa matrixmetalloproteinase-9. In arthritis patients, this enzyme can contributeto the pathogenesis of joint destruction and can be a useful marker ofdisease status.

Endogenous inhibitors of MMP-9. MMP-9 has a number of endogenousinhibitors. Like other MMPs, MMP-9 is inhibited by TIMPs (Murphy, G.,and Willenbrock, F. (1995) Methods Enzymol. 248, 496-510). Acharacteristic of MMP-9 (and MMP-2) is the ability of their zymogens toform tight non-covalent and stable complexes with TIMPs. It has beenshown that pro-MMP-2 binds TIMP-2 (Goldberg et al. (1989) Proc. Natl.Acad. Sci. U.S.A. 86, 8207-8211), whereas pro-MMP-9 binds TIMP-1(Wilhelm et al. (1989) J. Biol. Chem. 264, 17213-17221). TIMPs typicallyare slow, tight binding inhibitors. A MMP-9 binding protein (e.g.,antibody, peptide, Kunitz domain) selected from a library ofphage-displayed proteins can be selected have more rapid kinetics. Forexample, recombinant TIMP-1 can be administered to inhibit MMP-9, e.g.,in combination with a MMP-9 binding protein described herein.

Small molecule inhibitors of MMP-9. Skiles et al. (2004, Curr Med Chem,11:2911-77) reported that first generation small-molecule MMP inhibitorshad poor bioavailability and the second generation had causedmusculoskeletal pain and inflammation. Most small-molecule MMPinhibitors interact with the catalytic zinc but have fairly lowaffinity. Thus, a higher concentration is needed to have effect. Theinteraction with the catalytic zinc leads to inhibition of other MMPsand toxic side effects. A MMP-9 binding protein described herein can beused in combination with a small molecule inhibitor, For example,because the inhibitors are used in combination, the dose of the smallmolecule used can be decreased and therefore result in fewer sideeffects. Examples of small molecule MMP-9 inhibitors include smallsynthetic anthranilic acid-based inhibitors (see, e.g., CalbiochemInhibitor-I, catalogue #444278 and Levin et al., 2001, Bioorg. Med.Chem. Lett. 11:2975-2978).

Small interfering RNA inhibitors of MMP-9. MMP-9 can be inhibited bysmall interfering RNA (siRNA). Examples of siRNA that can be usedinclude:

MMP-9 siRNA (SEQ ID NO: 8) 5′-GACUUGCCGCGAGACAUGAtt-3′ (SEQ ID NO: 9)3′-ttCUGAACGGCGCUCUGUACU-5′ Control RNA (mismatch) (SEQ ID NO: 10)5′-GACUUCGCGGGACACAUGAtt-3′ (SEQ ID NO: 11) 3′-ttCUGAAGCGCCCUGUGUACU-5′

See also Kawasaki et al., Feb. 10, 2008, Nat. Med. advance on-linepublication doi:10.1038/nm1723. The siRNA can be administered to inhibitMMP-9, e.g., in combination with a MMP-9 binding protein describedherein.

MMP-9 Binding Proteins

Provided also are proteins that bind to MMP-9 (e.g., human MMP-9) andare either peptides, polypeptides that include at least one immunoglobinvariable region, or Kunitz domains. Methods for discovering andselecting and improving such binding proteins are described furtherbelow. MMP-9 expression is observed in bone samples from patients withbone-metastatic prostate cancer. An MMP-9 binding protein, e.g., anMMP-9 binding protein described herein, can be used in the methodsdescribed herein, e.g., to treat an osteolytic disorder.

In a preferred embodiment, the MMP-9 binding protein includes at leastone immunoglobulin variable domain. For example, the MMP-9 bindingprotein includes a heavy chain (HC) immunoglobulin variable domainsequence and a light chain (LC) immunoglobulin variable domain sequence.A number of exemplary MMP-9 binding proteins are described herein. MMP-9binding proteins may be antibodies. MMP-9 binding antibodies may havetheir HC and LC variable domain sequences included in a singlepolypeptide (e.g., scFv), or on different polypeptides (e.g., IgG orFab).

The MMP-9 binding protein may be an isolated peptide or protein (e.g.,at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% free ofother proteins).

The MMP-9 binding protein may additionally inhibit MMP-9, e.g., humanMMP-9. The binding protein can inhibit the catalytic activity of MMP-9(e.g., human MMP-9). In one embodiment, the protein binds the catalyticdomain of human MMP-9, e.g., the protein contacts residues in or nearthe active site of MMP-9. In some embodiments, the protein does notcontact residues in or near the active site of MMP-9 but instead bindselsewhere on MMP-9 and causes a steric change in MMP-9 that affects(e.g., inhibits) its activity.

The protein can bind to MMP-9, e.g., human MMP-9, with a bindingaffinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M⁻¹. In oneembodiment, the protein binds to MMP-9 with a K_(off) slower than1×10⁻³, 5×10⁻⁴ s⁻¹, or 1×10⁴ s⁻¹. In one embodiment, the protein bindsto MMP-12 with a K_(on) faster than 1×10², 1×10³, or 5×10³M⁻¹s⁻¹. In oneembodiment, the protein inhibits human MMP-9 activity, e.g., with a Kiof less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, and 10⁻¹° M. The protein canhave, for example, an 1050 of less than 100 nM, 10 nM or 1 nM. In someembodiments, the protein has an IC50 of about 1.8 nM. The affinity ofthe protein for MMP-9 can be characterized by a K_(D) of less than 100nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5 nM (e.g., 5nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM), or about 10nM (e.g., 9.6 nM).

In some embodiments, the protein has a K_(D)<200 nM.

In some embodiments, the protein has a t1/2 of at least about 10 minutes(e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), atleast about 25 minutes (e.g., 25 minutes), at least about 35 minutes(e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes).

In one embodiment, the protein binds the catalytic domain of humanMMP-9, e.g., the protein contacts residues in or near the active site ofMMP-9.

In some embodiments, the protein does not contact residues in or nearthe active site of MMP-9 but instead binds elsewhere on MMP-9 and causesa steric change in MMP-9 that affects (e.g., inhibits) its activity.

Exemplary MMP-9 binding proteins include antibodies with a heavy chain(HC) and/or light chain (LC), and in some embodiments, an HC and/or LCvariable domain, that is selected from the group of antibodiesconsisting of: 539A-M0240-B03, M0078-G07, M008′-D05, M0076-D03,M0072-H07, M0075-D12, and M0166-F10, or proteins that comprise the HCand/or LC CDRs of 539A-M0240-B03, M0078-G07, M0081-D05, M0076-D03,M0072-H07, M0075-D12, and M0166-F10. These MMP-9 binding proteins arefurther described in U.S. Ser. No. 61/033,075, filed Mar. 3, 2008 andU.S. Ser. No. 61/054,938, filed May 21, 2008, the content of whichapplications are hereby incorporated by reference in their entireties.Amino acid sequences for these and additional binding proteins are alsoprovided in FIG. 3 and the Examples herein.

The protein, if an antibody, can include one or more of the followingcharacteristics: (a) a human CDR or human framework region; (b) the HCimmunoglobulin variable domain sequence comprises one or more CDRs thatare at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to a CDR of a HC variable domain described above; (c) the LCimmunoglobulin variable domain sequence comprises one or more CDRs thatare at least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to a CDR of a LC variable domain described above; (d) the LCimmunoglobulin variable domain sequence is at least 85, 88, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domaindescribed above; (e) the HC immunoglobulin variable domain sequence isat least 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to a HC variable domain described above; (f) the protein bindsan epitope bound by a protein described herein, or an epitope thatoverlaps with such epitope; and (g) a primate CDR or primate frameworkregion.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain. In another, the HC and LCvariable domain sequences are components of different polypeptidechains. For example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, orIgG4. The protein can be a soluble Fab (sFab). In other implementationsthe protein includes a Fab₂′, scFv, minibody, scFv::Fc fusion, Fab::HSAfusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule thatcomprises the antigen combining site of one of the binding proteinsherein. The VH and VL regions of these Fabs can be provided as IgG, Fab,Fab₂, Fab₂′, scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab₂,VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, orother appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions. In one embodiment, the protein includes a human Fc domain, oran Fc domain that is at least 95, 96, 97, 98, or 99% identical to ahuman Fc domain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions. In one embodiment, the protein includes a primate Fc domain, oran Fc domain that is at least 95, 96, 97, 98, or 99% identical to aprimate Fe domain., “Primate” includes humans (Homo sapiens),chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas(Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentoniamadagascariensis), and tarsiers.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

MMP-9/MMP-2 Binding Proteins

MMP-9/2 binding proteins are binding proteins that bind to MMP-9 (e.g.,human MMP-9) and MMP-2 (e.g., human MMP-2) and are either peptides,polypeptides that include at least one immunoglobin variable region, orKunitz domains. Methods for discovering and selecting and improving suchbinding proteins are described further below. Both MMP-9 and MMP-2expression is observed in bone samples from patients withbone-metastatic prostate cancer. An MMP-9/MMP-2 binding protein, e.g.,an MMP-9/MMP-2 binding proteins described herein, can be used in themethods described herein, e.g., to treat an osteolytic disorder.

In a preferred embodiment, the MMP-9/2 binding protein includes at leastone immunoglobin variable region. For example, the MMP-9/MMP-2 bindingprotein includes a heavy chain (HC) immunoglobulin variable domainsequence and a light chain (LC) immunoglobulin variable domain sequence.MMP-9/MMP-2 binding proteins may be antibodies. MMP-9/MMP-2 bindingantibodies may have their HC and LC variable domain sequences includedin a single polypeptide (e.g., scFv), or on different polypeptides(e.g., IgG or Fab).

The MMP-9/MMP-2 binding protein may be an isolated protein (e.g., atleast 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% free ofother proteins).

The MMP-9/MMP-2 binding protein may additionally inhibit MMP-9, e.g.,human MMP-9 and/or MMP-2, e.g., human MMP-2. The binding protein caninhibit the catalytic activity of MMP-9 (e.g., human MMP-9) and/or MMP-2(e.g., human MMP-2). In one embodiment, the protein binds the catalyticdomain of human MMP-9, e.g., the protein contacts residues in or nearthe active site of MMP-9 and/or the protein binds the catalytic domainof human MMP-2, e.g., the protein contacts residues in or near theactive site of MMP-2. In some embodiments, the protein does not contactresidues in or near the active site of MMP-9 but instead binds elsewhereon MMP-9 and causes a steric change in MMP-9 that affects (e.g.,inhibits) its activity. In other embodiments, the protein does notcontact residues in or near the active site of MMP-2 but instead bindselsewhere on MMP-2 and causes a steric change in MMP-2 that affects(e.g., inhibits) its activity.

The protein can bind to MMP-9 and/or MMP-2 with a binding affinity of atleast 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹° and 10¹¹M⁻¹. In one embodiment, theprotein binds to MMP-9 and/or MMP-2 with a K_(off) slower than 1×10⁻³,5×10⁴ s⁻¹, or 1×10⁻⁴s⁻¹. In one embodiment, the protein binds to MMP-12with a K_(on) faster than 1×10², 1×10³, or 5×10³ M⁻¹s⁻¹. In oneembodiment, the protein inhibits human MMP-9 and/or MMP-2 activity,e.g., with a Ki of less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, and 10⁻¹⁰ M.The protein can have, for example, an IC₅₀ of less than 100 nM, 10 nM or1 nM. In some embodiments, the protein has an IC50 of about 1.8 nM. Theaffinity of the protein for MMP-9 can be characterized by a K_(D) ofless than 100 nm, less than 10 nM, or about 3 nM (e.g., 3.1 nM), about 5nM (e.g., 5 nM), about 6 nm (e.g., 5.9 nM), about 7 nM (e.g., 7.1 nM),or about 10 nM (e.g., 9.6 nM).

In some embodiments, the protein has a K_(D)<200 nM.

In some embodiments, the protein has a t1/2 of at least about 10 minutes(e.g., 11 minutes), at least about 20 minutes (e.g., 18 minutes), atleast about 25 minutes (e.g., 25 minutes), at least about 35 minutes(e.g., 33 minutes), or at least about 60 minutes (e.g., 57 minutes).

An exemplary MMP-9/2 binding protein includes an antibody with a heavychain (HC) and/or light chain (LC), and in some embodiments, an HCand/or LC variable domain, that is selected from the group of antibodiesconsisting of: M0237-D02. Such MMP-9/2 binding proteins are furtherdescribed in U.S. Ser. No. 61/033,068, filed on Mar. 3, 2008, U.S. Ser.No. 61/033,075, filed Mar. 3, 2008 and U.S. Ser. No. 61/054,938, filedMay 21, 2008, the content of which applications are hereby incorporatedby reference in their entireties. Amino acid sequences are also providedin the Examples herein.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain. In another, the HC and LCvariable domain sequences are components of different polypeptidechains. For example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, orIgG4. The protein can be a soluble Fab (sFab). In other implementationsthe protein includes a Fab₂′, scFv, minibody, scFv::Fc fusion, Fab::HSAfusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule thatcomprises the antigen combining site of one of the binding proteinsherein. The VH and VL regions of these Fabs can be provided as IgG, Fab,Fab₂, Fab₂′, scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab₂,VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, orother appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions. In one embodiment, the protein includes a human Fc domain, oran Fc domain that is at least 95, 96, 97, 98, or 99% identical to ahuman Fc domain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions. In one embodiment, the protein includes a primate Fc domain, oran Fc domain that is at least 95, 96, 97, 98, or 99% identical to aprimate Fc domain. “Primate” includes humans (Homo sapiens), chimpanzees(Pan troglodytes and Pan paniscus (bonobos)), gorillas (Gorillagorilla), gibons, monkeys, lemurs, aye-ayes (Daubentoniamadagascariensis), and tarsiers.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

Methods for Discovering MMP-14 or MMP-9 Binding Proteins

MMP-14 or MMP-9 binding proteins may be discovered by any method ofligand discovery known in the art. In certain embodiments, MMP-14 orMMP-9 binding proteins may be discovered by screening a library. Incertain embodiments, the library is a display library. A display libraryis a collection of entities; each entity includes an accessiblepolypeptide component and a recoverable component that encodes oridentifies the polypeptide component. The polypeptide component isvaried so that different amino acid sequences are represented. Thepolypeptide component can be of any length, e.g. from three amino acidsto over 300 amino acids. A display library entity can include more thanone polypeptide component, for example, the two polypeptide chains of asoluble Fab (sFab). In one exemplary implementation, a display librarycan be used to identify proteins that bind to MMP-14 or MMP-9. In aselection, the polypeptide component of each member of the library isprobed with MMP-14 or MMP-9 (e.g., the catalytic domain of MMP-14 orMMP-9 or other fragment) and if the polypeptide component binds to theMMP-14 or MMP-9, the display library member is identified, typically byretention on a support.

After selecting candidate library members that bind to a target, eachcandidate library member can be further analyzed, e.g., to furthercharacterize its binding properties for the target, e.g., MMP-14 orMMP-9, or for binding to another protein, e.g., anothermetalloproteinase. Each candidate library member can be subjected to oneor more secondary screening assays. The assay can be for a bindingproperty, a catalytic property, an inhibitory property, a physiologicalproperty (e.g., cytotoxicity, renal clearance, immunogenicity), astructural property (e.g., stability, conformation, oligomerizationstate) or another functional property. The same assay can be usedrepeatedly, but with varying conditions, e.g., to determine pH, ionic,or thermal sensitivities.

As appropriate, the assays can use a display library member directly, arecombinant polypeptide produced from the nucleic acid encoding theselected polypeptide, or a synthetic peptide synthesized based on thesequence of the selected polypeptide. In the case of selected Fabs, theFabs can be evaluated or can be modified and produced as intact IgGproteins. Exemplary assays for binding properties include ELISAs,homogenous binding assays, surface plasmon resonance (SPR) and cellularassays, the practice of which are well-known to those of skill in theart.

In addition to the use of display libraries, other methods can be usedto obtain a MMP-14 or MMP-9 binding antibody. For example, MMP-14 orMMP-9 protein or a region thereof can be used as an antigen in anon-human animal, e.g., a rodent. Humanized antibodies can be generatedby replacing sequences of the Fv variable region that are not directlyinvolved in antigen binding with equivalent sequences from human Fvvariable regions. General methods for generating humanized antibodiesare provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi etal., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos.5,585,089, 5,693,761 and 5,693,762. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable regions from at least one of aheavy or light chain, Numerous sources of such nucleic acid areavailable. For example, nucleic acids may be obtained from a hybridomaproducing an antibody against a predetermined target, as describedabove. The recombinant DNA encoding the humanized antibody, or fragmentthereof, can then be cloned into an appropriate expression vector.

Immunoglobin MMP-14 or MMP-9 binding proteins (e.g., IgG or Fab MMP-14or MMP-9 binding proteins) may be modified to reduce immunogenicity.Reduced immunogenicity is desirable in MMP-14 or MMP-9 binding proteinsintended for use as therapeutics, as it reduces the chance that thesubject will develop an immune response against the therapeuticmolecule. Techniques useful for reducing immunogenicity of MMP-14 orMMP-9 binding proteins include deletion/modification of potential humanT-cell epitopes and ‘germlining’ of sequences outside of the CDRs (e.g.,framework and Fc).

An MMP-14 or MMP-9-binding antibody may be modified by specific deletionof human T-cell epitopes or “deimmunization” by the methods disclosed inWO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variableregions of an antibody are analyzed for peptides that bind to MHC ClassII; these peptides represent potential T-cell epitopes (as defined in WO98/52976 and WO 00/34317). For detection of potential T-cell epitopes, acomputer modeling approach termed “peptide threading” can be applied,and in addition a database of human MHC class II binding peptides can besearched for motifs present in the VH and VL sequences, as described inWO 98/52976 and WO 00/34317. These motifs bind to any of the 18 majorMHC class II DR allotypes, and thus constitute potential T-cellepitopes. Potential T-cell epitopes detected can be eliminated bysubstituting small numbers of amino acid residues in the variableregions, or preferably, by single amino acid substitutions. As far aspossible conservative substitutions are made, often but not exclusively,an amino acid common at this position in human germline antibodysequences may be used. Human germline sequences are disclosed inTomlinson, I. A. et al., 1992, J Mol. Biol. 227:776-798; Cook, G. P. etal., 1995, Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al.,1992, J. Mol. Bio. 227:799-817. The V BASE directory provides acomprehensive directory of human immunoglobulin variable regionsequences (compiled by Tomlinson, I. A. et al. MRC Centre for ProteinEngineering, Cambridge, UK). After the deimmunizing changes areidentified, nucleic acids encoding V_(H) and V_(L) can be constructed bymutagenesis or other synthetic methods (e.g., de novo synthesis,cassette replacement, and so forth). Mutagenized variable sequence can,optionally, be fused to a human constant region, e.g., human IgG1 or κconstant regions.

In some cases a potential T-cell epitope will include residues which areknown or predicted to be important for antibody function. For example,potential T-cell epitopes are usually biased towards the CDRs. Inaddition, potential T-cell epitopes can occur in framework residuesimportant for antibody structure and binding. Changes to eliminate thesepotential epitopes will in some cases require more scrutiny, e.g., bymaking and testing chains with and without the change. Where possible,potential T-cell epitopes that overlap the CDRs were eliminated bysubstitutions outside the CDRs. In some cases, an alteration within aCDR is the only option, and thus variants with and without thissubstitution should be tested. In other cases, the substitution requiredto remove a potential T-cell epitope is at a residue position within theframework that might be critical for antibody binding. In these cases,variants with and without this substitution should be tested. Thus, insome cases several variant deimmunized heavy and light chain variableregions were designed and various heavy/light chain combinations testedin order to identify the optimal deimmunized antibody. The choice of thefinal deimmunized antibody can then be made by considering the bindingaffinity of the different variants in conjunction with the extent ofdeimmunization, i.e., the number of potential T-cell epitopes remainingin the variable region. Deimmunization can be used to modify anyantibody, e.g., an antibody that includes a non-human sequence, e.g., asynthetic antibody, a murine antibody other non-human monoclonalantibody, or an antibody isolated from a display library.

MMP-14 or MMP-9 binding antibodies are “germlined” by reverting one ormore non-germline amino acids in framework regions to correspondinggermline amino acids of the antibody, so long as binding properties aresubstantially retained. Similar methods can also be used in the constantregion, e.g., in constant immunoglobulin domains.

Antibodies that bind to MMP-14 or MMP-9, e.g., an antibody describedherein, may be modified in order, to make the variable regions of theantibody more similar to one or more germline sequences. For example, anantibody can include one, two, three, or more amino acid substitutions,e.g., in a framework, CDR, or constant region, to make it more similarto a reference germline sequence. One exemplary germlining method caninclude identifying one or more germline sequences that are similar(e.g., most similar in a particular database) to the sequence of theisolated antibody. Mutations (at the amino acid level) are then made inthe isolated antibody, either incrementally or in combination with othermutations. For example, a nucleic acid library that includes sequencesencoding some or all possible germline mutations is made. The mutatedantibodies are then evaluated, e.g., to identify an antibody that hasone or more additional germline residues relative to the isolatedantibody and that is still useful (e.g., has a functional activity). Inone embodiment, as many germline residues are introduced into anisolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a framework and/or constant region. Forexample, a germline framework and/or constant region residue can be froma germline sequence that is similar (e.g., most similar) to thenon-variable region being modified. After mutagenesis, activity (e.g.,binding or other functional activity) of the antibody can be evaluatedto determine if the germline residue or residues are tolerated (i.e., donot abrogate activity). Similar mutagenesis can be performed in theframework regions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may include usingtwo germline sequences that separately contribute to the amino-terminalportion and the carboxy-terminal portion. In other implementations morethan one or two germline sequences are used, e.g., to form a consensussequence.

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% ofthe CDR amino acid positions that are not identical to residues in thereference CDR sequences, residues that are identical to residues atcorresponding positions in a human germline sequence (i.e., an aminoacid sequence encoded by a human germline nucleic acid).

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 50, 60, 70, 80, 90 or 100% of the FRregions identical to FR sequence from a human germline sequence, e.g., agermline sequence related to the reference variable domain sequence.

Accordingly, it is possible to isolate an antibody which has similaractivity to a given antibody of interest, but is more similar to one ormore germline sequences, particularly one or more human germlinesequences. For example, an antibody can be at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 99.5% identical to a germline sequence in aregion outside the CDRs (e.g., framework regions). Further, an antibodycan include at least 1, 2, 3, 4, or 5 germline residues in a CDR region,the germline residue being from a germline sequence of similar (e.g.,most similar) to the variable region being modified. Germline sequencesof primary interest are human germline sequences. The activity of theantibody (e.g., the binding activity as measured by K_(A)) can be withina factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.

Germline sequences of human immunoglobin genes have been determined andare available from a number of sources, including the internationalImMunoGeneTics information system (IMGT), available via the world wideweb at imgt.cines.fr, and the V BASE directory (compiled by Tomlinson,I. A. et al. MRC Centre for Protein Engineering, Cambridge, UK,available via the world wide web at vbase.mrc-cpe.cam.ac.uk).

Exemplary germline reference sequences for V_(kappa) include: O12/O2,O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23, L9, L24, L11,L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11, L2/L16, L6, L20,L25, B3, B2, A26/A10, and A14. See, e.g., Tomlinson et al., 1995, EMBOJ. 14(18):4628-3.

A germline reference sequence for the HC variable domain can be based ona sequence that has particular canonical structures, e.g., 1-3structures in the H1 and H2 hypervariable loops. The canonicalstructures of hypervariable loops of an immunoglobulin variable domaincan be inferred from its sequence, as described in Chothia et al., 1992,J. Mol. Biol. 227:799-817; Tomlinson et al., 1992, J. Mol. Biol.227:776-798); and Tomlinson et al., 1995, EMBO J. 14(18):4628-38.Exemplary sequences with a 1-3 structure include: DP-1, DP-8, DP-12,DP-2, DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2,hv3005, hv3005f3, DP-46, DP-47, DP-58, DP-49, DP-50, DP-51, DP-53, andDP-54.

In one embodiment, an MMP-14 or MMP-9 binding protein is physicallyassociated with a moiety that improves its stabilization and/orretention in circulation, e.g., in blood, serum, lymph, or othertissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, anMMP-14 or MMP-9 binding protein can be associated with a polymer, e.g.,a substantially non-antigenic polymers, such as polyalkylene oxides orpolyethylene oxides. Suitable polymers will vary substantially byweight. Polymers having molecular number average weights ranging fromabout 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 toabout 12,500) can be used. For example, an MMP-14 or MMP-9 bindingprotein can be conjugated to a water soluble polymer, e.g., hydrophilicpolyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. Anon-limiting list of such polymers include polyalkylene oxidehomopolymers such as polyethylene glycol (PEG) or polypropylene glycols,polyoxyethylenated polyols, copolymers thereof and block copolymersthereof, provided that the water solubility of the block copolymers ismaintained.

An MMP-14 or MMP-9 binding protein can also be associated with a carrierprotein, e.g., a serum albumin, such as a human serum albumin. Forexample, a translational fusion can be used to associate the carrierprotein with the MMP-14 or MMP-9 binding protein.

Pharmaceutical Compositions of MMP-14 or MMP-9 Binding Proteins

In another aspect, the disclosure provides compositions, e.g.,pharmaceutically acceptable compositions or pharmaceutical compositions,which include an MMP-14 or MMP-9-binding protein, e.g., an antibodymolecule, other polypeptide or peptide identified as binding to MMP-14or MMP-9 described herein. The MMP-14 or MMP-9 binding protein can beformulated together with a pharmaceutically acceptable carrier and/orpharmaceutically acceptable salt. Pharmaceutical compositions includetherapeutic compositions and diagnostic compositions, e.g., compositionsthat include labeled MMP-14 or MMP-9 binding proteins for in vivoimaging.

A pharmaceutically acceptable carrier includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal, orepidermal administration (e.g., by injection or infusion), althoughcarriers suitable for inhalation and intranasal administration are alsocontemplated. Depending on the route of administration, the MMP-14 orMMP-9 binding protein may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

A pharmaceutically acceptable salt is a salt that retains the desiredbiological activity of the parent compound and does not impart anyundesired toxicological effects (see e.g., Berge, S. M., et al., 1977,J. Pharm. Sci. 66:1-19). Examples of such salts include acid additionsalts and base addition salts. Acid addition salts include those derivedfrom nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well asfrom nontoxic organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,aromatic acids, aliphatic and aromatic sulfonic acids, and the like.Base addition salts include those derived from alkaline earth metals,such as sodium, potassium, magnesium, calcium, and the like, as well asfrom nontoxic organic amines, such as N,N′-dibenzylethylenediamine,N-methylglucamine, chloroprocaine, choline, diethanolamine,ethylenediamine, procaine, and the like.

The compositions may be in a variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform can depend on the intended mode of administration and therapeuticapplication. Many compositions are in the form of injectable orinfusible solutions, such as compositions similar to those used foradministration of humans with antibodies. An exemplary mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one embodiment, the MMP-14 or MMP-9binding protein is administered by intravenous infusion or injection. Inanother preferred embodiment, the MMP-14 or MMP-9 binding protein isadministered by intramuscular or subcutaneous injection.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the binding protein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

An MMP-14 or MMP-9 binding protein can be administered by a variety ofmethods, although for many applications, the preferred route/mode ofadministration is intravenous injection or infusion. For example, fortherapeutic applications, the MMP-14 or MMP-9 binding protein can beadministered by intravenous infusion at a rate of less than 30, 20, 10,5, or 1 mg/min to reach a dose of about 1 to 100 mg/m² or 7 to 25 mg/m².The route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are available. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., 1978, Marcel Dekker, Inc., New York.

Pharmaceutical compositions can be administered with medical devices.For example, in one embodiment, a pharmaceutical composition disclosedherein can be administered with a device, e.g., a needleless hypodermicinjection device, a pump, or implant.

In certain embodiments, an MMP-14 or MMP-9 binding protein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds disclosed herein cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties that are selectively transported into specific cells or organs,thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, J.Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms can be dictated by and directly dependent on(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody disclosed herein is0.1-20 mg/kg, more preferably 1-10 mg/kg. An anti-MMP-14 or MMP-9antibody can be administered, e.g., by intravenous infusion, e.g., at arate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1to 100 mg/m² or about 5 to 30 mg/m². For binding proteins smaller inmolecular weight than an antibody, appropriate amounts can beproportionally less. Dosage values may vary with the type and severityof the condition to be alleviated. For a particular subject, specificdosage regimens can be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions.

The pharmaceutical compositions disclosed herein may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an MMP-14 or MMP-9 binding protein disclosed herein.

Methods of Treating Osteolytic Disorders

Proteins that bind to MMP-14 or MMP-9 and identified by the methoddescribed herein and/or detailed herein have therapeutic andprophylactic utilities, particularly in human subjects. These bindingproteins are administered to a subject to treat, prevent, and/ordiagnose osteolytic disorders. In certain embodiments, the MMP-14 orMMP-9 binding proteins are administered to a subject, or even toosteotropic cancer cells in culture, e.g. in vitro or ex vivo, to treator prevent osteotropic cancer. In other embodiments, the MMP-14 or MMP-9binding proteins are administered to a subject to treat or preventosteoporosis. Treating includes administering an amount effective toalleviate, relieve, alter, remedy, ameliorate, improve or affect thedisorder, at least one symptom of the disorder or the predispositiontoward the disorder, whereas preventing includes administering an amounteffective to stop or slow the manifestation of the disorder, e.g., ascompared to what is expected in the absence of the treatment. Thetreatment may also delay onset, e.g., prevent onset, or preventdeterioration of the osteolytic disorder, e.g., as compared to what isexpected in the absence of the treatment.

As used herein, an amount of a MMP-14 or MMP-9 binding protein effectiveto prevent an osteolytic disorder, such as osteotropic cancer orosteoporosis, or a prophylactically effective amount of the MMP-14 orMMP-9 binding protein, refers to an amount of a MMP-14 or MMP-9 bindingprotein, e.g., an anti-MMP-14 or MMP-9 antibody described herein, whichis effective, upon single- or multiple-dose administration to thesubject, for preventing or delaying the occurrence of the onset orrecurrence of the osteolytic disorder. Stated another way, atherapeutically effective amount of an MMP-14 or MMP-9 binding proteinis the amount which is effective, upon single or multiple doseadministration to a subject, in treating a subject, e.g., curing,alleviating, relieving or improving at least one symptom of a disorderin a subject to a degree beyond that expected in the absence of suchtreatment. A therapeutically effective amount of the composition mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the compound to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the compositionare outweighed by the therapeutically beneficial effects. Atherapeutically effective dosage preferably modulates a measurableparameter favorably relative to untreated subjects. The ability of acompound to inhibit a measurable parameter can be evaluated in an animalmodel system predictive of efficacy in a human disorder. Dosage regimenscan be adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Guidance for determination of a therapeutically effective amount fortreatment of an osteolytic disorder may be obtained by reference to invivo models of the particular osteolytic disorder. For example, forosteotropic cancer, the amount of a MMP-14 or MMP-9 binding protein thatis a therapeutically effective amount in a rodent or Libechov minipigmodel of cancer may be used to guide the selection of a dose that is atherapeutically effective amount. A number of rodent models of humancancers are available, including nude mouse/tumor xenograft systems.Cancer cell lines such as PC-3 or the human breast cancer cell line,MDA-MB-231, with either a high potential to cause bone metastasis(MDA-231#16) or a low potential (MDA-MB-231#17), may be used in thepreparation of such animal models, or may be used on their own asmodels.

A MMP-14 or MMP-9 binding protein described herein can be used to reducean osteolytic disorder in a subject, e.g., to treat an osteotropiccancer (e.g., a solid tumor or lesion, or to kill circulating cancercells) or osteoporosis (e.g., to reduce the porosity of the bones). Themethod includes administering the MMP-14 or MMP-9 binding protein to thesubject, e.g., in an amount effective to modulate the osteolyticdisorder (e.g., for osteotropic cancer, a tumor or lesion size), asymptom of the disorder, or progression of the disorder. The MMP-14 orMMP-9 binding protein may be administered multiple times (e.g., at leasttwo, three, five, or ten times) before a therapeutically effectiveamount is attained. In one embodiment, the MMP-14 or MMP-9 bindingproteins are used to inhibit an activity (e.g., inhibit at least oneactivity, reduce proliferation, migration, growth or viability) of acell, e.g., a cancer cell in vivo. The binding proteins can be used bythemselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxinenzyme, or radioisotope. This method includes: administering the bindingprotein alone or attached to an agent (e.g., a cytotoxic drug), to asubject requiring such treatment. For example, MMP-14 or MMP-9 bindingproteins that do not substantially inhibit MMP-14 or MMP-9 may be usedto deliver nanoparticles containing agents, such as toxins, to MMP-14 orMMP-9 associated cells or tissues, e.g., tumors.

Accordingly, the disclosure provides methods of treating (e.g., slowing,eliminating, or reversing tumor growth or bone porosity, preventing orreducing, either in number or size, metastases, reducing or eliminatingtumor cell invasiveness, providing an increased interval to tumorprogression, or increasing disease-free survival time, e.g., relative toa standard, e.g., as compared to what is expected in the absence oftreatment or as compared to the condition of a subject (or cohort ofsubjects) with an osteolytic disorder that was not treated for thedisease) an osteolytic disorder such as osteotropic cancer orosteoporosis by administering an effective amount of an MMP-14 or MMP-9binding protein (e.g., an anti-MMP-14 or MMP-9 IgG or Fab). In someembodiments, the MMP-14 or MMP-9 binding protein inhibits MMP-14 orMMP-9 activity. In certain embodiments, the MMP-14 or MMP-9 bindingprotein is administered as a single agent treatment. In otherembodiments, the MMP-14 or MMP-9 binding protein is administered incombination with an additional anti-cancer agent.

Also provided are methods of preventing or reducing risk of developingan osteolytic disorder, by administering an effective amount of anMMP-14 or MMP-9 binding protein to a subject at risk of developing anosteolytic disorder, thereby reducing the subject's risk of developingthe osteolytic disorder. For example, MMP-14 or MMP-9 binding proteinsmay be administered to prevent osteolytic lesions in a subject havingosteotropic cancer, e.g., bone metastasis. As another example, toprevent or reduce the risk of developing an osteolytic disorder, anMMP-14 or MMP-9 binding protein may be administered to a subject who hasbeen diagnosed with a cancer (e.g., breast, lung or prostate cancer)that has the potential to metastasize to bone.

The disclosure further provides methods of modulating (e.g., reducing orpreventing) osteotropic cancer at a tumor site by administering aneffective amount of an MMP-14 or MMP-9 binding protein, thereby reducingor preventing the tumor size or growth. The MMP-14 or MMP-9 bindingprotein may be administered to the tumor site as a single agent therapyor in combination with additional agents.

Also provided are methods for reducing extracellular matrix (ECM)degradation by a tumor, comprising administering an effective amount ofan MMP-14 or MMP-9 binding protein to a subject, thereby reducing ECMdegradation by a tumor in the subject.

Methods of administering MMP-14 or MMP-9 binding proteins and otheragents are also described in “Pharmaceutical Compositions.” Suitabledosages of the molecules used can depend on the age and weight of thesubject and the particular drug used. The binding proteins can be usedas competitive agents to inhibit, reduce an undesirable interaction,e.g., between a natural or pathological agent and the MMP-14 or MMP-9.The dose of the MMP-14 or MMP-9 binding protein can be the amountsufficient to block 90%, 95%, 99%, or 99.9% of the activity of MMP-14 orMMP-9 in the patient, especially at the site of disease. Depending onthe disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg/Kg. For anIgG having a molecular mass of 150,000 g/mole (two binding sites), thesedoses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and1.8 μM of binding sites for a 5 L blood volume.

Because the MMP-14 or MMP-9 binding proteins recognize MMP-14 orMMP-9-expressing cells and can bind to cells that are associated with(e.g., in proximity of or intermingled with) osteotropic cancer cells,MMP-14 or MMP-9 binding proteins can be used to inhibit (e.g., inhibitat least one activity, reduce growth and proliferation, or kill) anysuch cells and inhibit the progression of the osteolytic disorder.Reducing MMP-14 or MMP-9 activity near a cancer can indirectly inhibit(e.g., inhibit at least one activity, reduce growth and proliferation,or kill) the cancer cells which may be dependent on the MMP-14 or MMP-9activity for metastasis, activation of growth factors, and so forth.

Alternatively, the binding proteins bind to cells in the vicinity of thecancerous cells, but are sufficiently close to the cancerous cells todirectly or indirectly inhibit (e.g., inhibit at least one activity,reduce growth and proliferation, or kill) the cancers cells. Thus, theMMP-14 or MMP-9 binding proteins (e.g., modified with a toxin, e.g., acytotoxin) can be used to selectively inhibit cells in cancerous tissue(including the cancerous cells themselves and cells associated with orinvading the cancer).

The MMP-14 or MMP-9 binding proteins may be used to deliver or aid orenhance the delivery of an agent (e.g., any of a variety of cytotoxicand therapeutic drugs) to cells and tissues where MMP-14 or MMP-9 ispresent. Exemplary agents include a compound emitting radiation,molecules of plants, fungal, or bacterial origin, biological proteins,and mixtures thereof. The cytotoxic drugs can be intracellularly actingcytotoxic drugs, such as toxins or short range radiation emitters, e.g.,short range, high energy α-emitters.

To target MMP-14 or MMP-9 expressing osteotropic cancer cells, a prodrugsystem can be used. For example, a first binding protein is conjugatedwith a prodrug which is activated only when in close proximity with aprodrug activator. The prodrug activator is conjugated with a secondbinding protein, preferably one which binds to a non competing site onthe target molecule. Whether two binding proteins bind to competing ornon competing binding sites can be determined by conventionalcompetitive binding assays. Exemplary drug prodrug pairs are describedin Blakely et al., (1996) Cancer Research, 56:3287 3292.

The MMP-14 or MMP-9 binding proteins can be used directly in vivo toeliminate antigen-expressing cells via natural complement-dependentcytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).The binding proteins described herein can include a complement bindingeffector domain, such as the Fc portions from IgG1, -2, or -3 orcorresponding portions of IgM which bind complement. In one embodiment,a population of target cells is ex vivo treated with a binding agentdescribed herein and appropriate effector cells. The treatment can besupplemented by the addition of complement or serum containingcomplement. Further, phagocytosis of target cells coated with a bindingprotein described herein can be improved by binding of complementproteins. In another embodiment, target cells coated with the bindingprotein which includes a complement binding effector domain are lysed bycomplement.

The MMP-14 or MMP-9 binding protein can be used to deliver macro andmicromolecules, e.g., a gene into the cell for gene therapy purposesinto the endothelium or epithelium and target only those tissuesexpressing the MMP-14 or MMP-9.

In the case of polypeptide toxins, recombinant nucleic acid techniquescan be used to construct a nucleic acid that encodes the binding protein(e.g., antibody or antigen-binding fragment thereof) and the cytotoxin(or a polypeptide component thereof) as translational fusions. Therecombinant nucleic acid is then expressed, e.g., in cells and theencoded fusion polypeptide isolated.

Alternatively, the MMP-14 or MMP-9 binding protein can be coupled tohigh energy radiation emitters, for example, a radioisotope, such as¹³¹I, a γ-emitter, which, when localized at a site, results in a killingof several cell diameters. See, e.g., S.E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303 316 (Academic Press 1985).Other suitable radioisotopes include a emitters, such as ²¹²Bi, ²¹³Bi,and ²¹¹At, and b emitters, such as ¹⁸⁶Re and ⁹⁰Y. Moreover, ¹⁷⁷Lu mayalso be used as both an imaging and cytotoxic agent.

Radioimmunotherapy (RIT) using antibodies labeled with ¹³¹I, ⁹⁰Y, and¹⁷⁷Lu is under intense clinical investigation. There are significantdifferences in the physical characteristics of these three nuclides andas a result, the choice of radionuclide is very critical in order todeliver maximum radiation dose to a tissue of interest. The higher betaenergy particles of ⁹⁰Y may be good for bulky tumors. The relatively lowenergy beta particles of ¹³¹I are ideal, but in vivo dehalogenation ofradioiodinated molecules is a major disadvantage for internalizingantibody. In contrast, ¹⁷⁷Lu has low energy beta particle with only0.2-0.3 mm range and delivers much lower radiation dose to bone marrowcompared to ⁹⁰Y. In addition, due to longer physical half-life (comparedto ⁹⁰Y), the residence times are higher. As a result, higher activities(more mCi amounts) of ¹⁷⁷Lu labeled agents can be administered withcomparatively less radiation dose to marrow. There have been severalclinical studies investigating the use of ¹⁷⁷Lu labeled antibodies inthe treatment of various cancers. (Mulligan T et al., 1995, Clin. Canc.Res. 1: 1447-1454; Meredith R F, et al., 1996, J. Nucl. Med.37:1491-1496; Alvarez R D, et al., 1997, Gynecol. Oncol. 65: 94-101).

Combination Therapies

The MMP-14 or MMP-9 binding proteins described herein, e.g., anti-MMP-14or MMP-9 Fabs or IgGs, can be administered in combination with one ormore of the other therapies for treating the particular osteolyticdisorder of interest. For example, an MMP-14 or MMP-9 binding proteincan be used therapeutically or prophylactically with surgery, anotherMMP-14 or MMP-9 inhibitor, e.g., a small molecule inhibitor, anotheranti-MMP-14 or MMP-9 Fab or IgG (e.g., another Fab or IgG describedherein), peptide inhibitor, or small molecule inhibitor. Examples ofMMP-14 or MMP-9 inhibitors that can be used in combination therapy withan MMP-14 or MMP-9 binding protein described herein include neovastat,marimastat, BAY 12-9566 and prinomastat. One or more small-molecule MMPinhibitors can be used in combination with one or more MMP-14 or MMP-9binding proteins described herein. For example, the combination canresult in a lower dose of the small-molecule inhibitor being needed,such that side effects are reduced. The combination may result inenhanced delivery and efficacy of one or both agents.

In certain embodiments, the MMP-14 or MMP-9 binding proteins describedherein can be administered in combination with one or more of the othertherapies for treating osteotropic cancer, including, but not limitedto: surgery; radiation therapy, chemotherapy, and other anti-cancertherapeutic agents. For example, proteins that inhibit MMP-14 or MMP-9or that inhibit a downstream event of MMP-14 or MMP-9 activity (e.g.,cleavage of pro-MMP-2 to MMP-2) can also be used in combination withother anti-cancer therapies, such as radiation therapy, chemotherapy,surgery, or administration of a second agent. For example, the secondagent can be a Tie-1 inhibitor (e.g., Tie-1 binding proteins; see e.g.,U.S. Ser. No. 11/199,739 and PCT/US2005/0284, both filed Aug. 9, 2005).As another example, the second agent can be one that targets ornegatively regulates the VEGF signaling pathway. Examples of this latterclass include VEGF antagonists (e.g., anti-VEGF antibodies such asbevacizumab) and VEGF receptor antagonists (e.g., anti-VEGF receptorantibodies). One particularly preferred combination includesbevacizumab. The combination can further include 5-FU and leucovorin,and/or irinotecan. Other additional cancer therapeutic or treatments,include bisphosphonates (e.g., amino and non-amino bisphosphonates),hormone-related compounds (e.g., estrogens and SERMs), RANKLantagonists, RANKL pathway inhibitors, α_(γ)β₃ antagonists, Srcinhibitors, cathepsin K inhibitors and calcitonin.

In other embodiments, the MMP-14 or MMP-9 binding proteins describedherein can be administered in combination with one or more of the othertherapies for treating osteoporosis, including, but not limited to,bisphosphonates (e.g., amino and non-amino bisphosphonates),hormone-related compounds (e.g., estrogens and SERMs), calcitonin,Teriparatide (FORTEO™), tamoxifen and RANKL pathway inhibitors.

The agents or therapies can be administered at the same time (e.g., as asingle formulation that is administered to a patient or as two separateformulations administered concurrently) or sequentially in any order.Sequential administrations are administrations that are given atdifferent times. The time between administration of the one agent andanother agent can be minutes, hours, days, or weeks. The use of anMMP-14 or MMP-9 binding protein described herein can also be used toreduce the dosage of another therapy, e.g., to reduce the side-effectsassociated with another agent that is being administered, e.g., toreduce the side-effects of an anti-VEGF antibody such as bevacizumab.Accordingly, a combination can include administering a second agent at adosage at least 10, 20, 30, or 50% lower than would be used in theabsence of the MMP-14 or MMP-9 binding protein.

The second agent or therapy can also be another anti-cancer agent ortherapy. Non-limiting examples of anti-cancer agents include, e.g.,anti-microtubule agents, topoisomerase inhibitors, antimetabolites,mitotic inhibitors, alkylating agents, intercalating agents, agentscapable of interfering with a signal transduction pathway, agents thatpromote apoptosis, radiation, and antibodies against othertumor-associated antigens (including naked antibodies, immunotoxins andradioconjugates). Examples of the particular classes of anti-canceragents are provided in detail as follows: antitubulin/antimicrotubule,e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere; topoisomerase I inhibitors, e.g., irinotecan, topotecan,camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin,idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantronehydrochloride; antimetabolites, e.g., 5 fluorouracil (5 FU),methotrexate, 6 mercaptopurine, 6 thioguanine, fludarabine phosphate,cytarabine/Ara C, trimetrexate, gemcitabine, acivicin, alanosine,pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA, pentostatin, 5azacitidine, 5 Aza 2′ deoxycytidine, ara A, cladribine, 5 fluorouridine,FUDR, tiazofurin, N-[5-N-(3,4-dihydro-2-methyl-4-oxoquinazolin-to6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid; alkylatingagents, e.g., cisplatin, carboplatin, mitomycin C, BCNU=Carmustine,melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine,ifosfamide phosphate, cyclophosphamide, nitrogen mustard, uracilmustard, pipobroman, 4 ipomeanol; agents acting via other mechanisms ofaction, e.g., dihydrolenperone, spiromustine, and desipeptide;biological response modifiers, e.g., to enhance anti-tumor responses,such as interferon; apoptotic agents, such as actinomycin D; andanti-hormones, for example anti-estrogens such as tamoxifen or, forexample anti-androgens such as4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide.

Other additional cancer therapeutic or treatments that may be used intreating metastatic bone cancers include bisphosphonates (e.g., aminoand non-amino bisphosphonates), hormone-related compounds (e.g.,estrogens and SERMs), RANKL antagonists, α_(γ)β₃ antagonists, Srcinhibitors, cathepsin K inhibitors and calcitonin. All of thesetherapeutics may serve as bone resorption inhibitors, in addition tohaving other activities.

Bisphosphonates (also called: diphosphonates) are a class of drugs thatinhibits osteoclast action and the resorption of bone. Their usesinclude the prevention and treatment of osteoporosis, osteitis deformans(“Paget's disease of bone”), bone metastasis (with or withouthypercalcemia), multiple myeloma and other conditions that feature bonefragility. Exemplary bisphosphonates (also known as diphosphonates)include both amino and non-amino bisphosphonates. Specific examples ofbisphosphonates that may be used in the disclosed methods include, butare not limited to, non-amino bisphosphonates such as Etidronate(DIDRONEL®), Clodronate (BONEFOS®, LORON®) and Tiludronate (SKELID®);and amino bisphosphonates such as Pamidronate (APD,AREDIA®),Neridronate, Olpadronate, Alendronate (FOSAMAX®), Ibandronate (BONIVA®),Risedronate (ACTONEL®) and Zoledronate (ZOMETA®).

Hormone-related compounds include, but are not limited to, estrogens,selective estrogen receptor modulators (SERMs) and LH-RH agonists suchas Leuprolide (LUPRON®, VIADUR®, ELIGARD®), Goserelin (Zoladex®),Raloxifene (EVISTA®) and Triptorelin (TRELSTAR®).

RANKL antagonists may be used to block RANK-RANKL interactions.Exemplary RANKL antagonists include, but are not limited to, TRANCE-Fc,OPG and OPG-Fc.

Exemplary RANKL pathway inhibitors include, but are not limited to,Denosumab (Body, et al. (2006) Clin. Cancer Res. 12:1221-1228).

α_(γ)β₃ antagonists may be used to block osteoclast adhesion to bone.Exemplary α_(γ)β₃ antagonists include small molecule and peptideantagonists, examples of which include but are not limited to, Vitaxin,Cilengitide,(S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-aceticacid and3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)propyl]imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)propionicacid.

Src inhibitors may be used to block steps leading to osteoclastactivation. Exemplary Src inhibitors include, but are not limited to,SKI-606 (Wyeth), AZD0530 (AstraZeneca) and BMS-453825 (Dasatinib(SPRYCEL®)).

Cathepsin K inhibitors may be used to block activity ofosteoclast-specific collagenase. Exemplary cathepsin K inhibitorsinclude, but are not limited to, balicatib.

A combination therapy can include administering an agent that reducesthe side effects of other therapies. In embodiments where the osteolyticdisorder is osteotropic cancer, the agent can be an agent that reducesthe side effects of anti-cancer treatments. For example, the agent canbe leucovorin.

Kits

An MMP-14 or MMP-9 binding protein described herein can be provided in akit, e.g., as a component of a kit. For example, the kit includes (a) anMMP-14 or MMP-9 binding protein, e.g., a composition that includes anMMP-14 or MMP-9 binding protein, and, optionally (b) informationalmaterial. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of an MMP-14 or MMP-9 binding protein for the methodsdescribed herein.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tousing the binding protein to treat, prevent, or diagnose an osteolyticdisorder.

In one embodiment, the informational material can include instructionsto administer an MMP-14 or MMP-9 binding protein in a suitable manner toperform the methods described herein, e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer an MMP-14or MMP-9 binding protein to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for, an osteolytic disorder. For example, thematerial can include instructions to administer an MMP-14 or MMP-9binding protein to a patient with osteotropic cancer or osteoporosis.The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin print but may also be in other formats, such as computer readablematerial.

An MMP-14 or MMP-9 binding protein can be provided in any form, e.g.,liquid, dried or lyophilized form. It is preferred that an MMP-14 orMMP-9 binding protein be substantially pure and/or sterile. When anMMP-14 or MMP-9 binding protein is provided in a liquid solution, theliquid solution preferably is an aqueous solution, with a sterileaqueous solution being preferred. When an MMP-14 or MMP-9 bindingprotein is provided as a dried form, reconstitution generally is by theaddition of a suitable solvent. The solvent, e.g., sterile water orbuffer, can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining an MMP-14 or MMP-9 binding protein. In some embodiments, thekit contains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in association with the container. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of anMMP-14 or MMP-9 binding protein. For example, the kit includes aplurality of syringes, ampules, foil packets, or blister packs, eachcontaining a single unit dose of an MMP-14 or MMP-9 binding protein. Thecontainers of the kits can be air tight, waterproof (e.g., impermeableto changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, dropper (e.g., eye dropper),swab (e.g., a cotton swab or wooden swab), or any such delivery device.In one embodiment, the device is an implantable device that dispensesmetered doses of the binding protein. The disclosure also features amethod of providing a kit, e.g., by combining components describedherein.

EXEMPLIFICATIONS

The following examples provide further illustration and are notlimiting.

Example 1 DX-2400 Reduces Osteolytic Lesions in the PC-3 Prostate CancerModel

PC-3 prostate cancer cells were inoculated intra-tibially into mice.Treatment was initiated 3 days after intra-tibial inoculation of thecells. DX-2400 (10 mg/kg) and a PBS control were administered Q2D for 14days. X-ray analysis and bone histomorphometric analysis indicated thatDX-2400 reduced the area of the osteolytic lesions about 3-fold (FIG.2). DX-2400 is a selective inhibitor of MMP-14.

Example 2 Exemplary MMP-9 Binding Antibodies

Experiments were performed to evaluate the in vitro effects of539A-M0240-B03 and 539A-M0237-D02 in bone metastasis models.

Cells. Raw 264.7 cells (Mouse leukemia monocyte/macrophage cell line)were obtained from ATCC (Catalog #TIB-71) and maintained in ATCCrecommended complete medium (Catalog #30-2020). Cells between passage3-7 were used in this study.

Materials. Osteologic discs were purchased from BD biosciences (Catalog#354609). Tartrate-resistant acid phosphatase (TRAP) staining kit wasobtained from Kamiya Biomedica Company, Seattle Wash. (Catalog #KT-008).GM6001 was obtained from Millipore.

Methods. Approximately 2000 Raw 264.7 cells per slide were seeded ontoosteologic multitest slides with complete growth medium. On thefollowing day, cells were replaced with fresh medium containing 100ng/ml recombinant murine soluble RANK ligand (Peprotech Inc. UK) alongwith the broad spectrum MMP inhibitor GM6001 (5 μM, 10 μM, 25 μMconcentrations tested), 539A-M0240-B03 (10 μg/ml, 50 μg/mlconcentrations tested), or 539A-M0237-D02 (10 μg/ml, 100 μg/mlconcentrations tested). The slides were then incubated at 37° C. for 6days, replacing fresh media on day 3 as described above. At the end ofincubation time, one side of the slide was stained for TRAP and theother side of the slide was bleached (10% bleach), washed several timeswith water, and air dried. The slides were then viewed under themicroscope for either multinucleated TRAP positive cells or resorbedareas (pits). Cells incubated with media only and recombinantosteoprotegerin (rH OPG) (100 ng/ml) served as negative and positivecontrols, respectively.

Conclusion. These in vitro experiments suggest that GM6001,539A-MO240-B03, and 539A-M0237D02 have inhibitory effects onosteoclastogenesis and bone resorption at the concentrations tested(data not shown). The results showed that the inhibitory effect wasdose-dependent. As the concentration of GM6001, 539A-M0240-B03, or539A-M0237D02 increased, the amount of TRAP-positive staining and thenumber of resorbed areas decreased.

Example 3 Exemplary MMP-9 Binding Antibodies

539A-M0166-F10. An exemplary MMP-9 antibody is 539A-M0166-F10. The aminoacid sequences of variable regions of 539A-M0166-F10 sFAB are asfollows:

539A-M0166-F10 (phage/SFAB) VL leader + VL (SEQ ID NO: 12)FYSHSAQSELTQPPSASAAPGQRVTISCSGSSSNIGSNTVTWYQKLPGTAPKLLIYNNYERPSGVPARFSGSKSGTSASLAISGLQSEDEADYYCATWDDSLIANYVFGSGTKVTVLGQPKANP  539A-M0166-F10 (phage/SFAB) VH leader + VH(SEQ ID NO: 13) MKKLLFAIPLVVPFVAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSPYLMNWVRQAPGKGLEWVSSIYSSGGGTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIYHSSSGPFYGMDVWGQGTTVTVSSAST KGPSVFPLAPSSKS 

539A-M0240-B03. Another exemplary MMP-9 antibody is 539A-M0240-B03.539A-M0240-B03 is a selective inhibitor of MMP-9. 539A-M0240-B03 candecrease or inhibit the activity of human and mouse MMP-9. The sequencesof the complememtarity determining regions (CDRs) of 539A-M0240-B03light chain (LC) and heavy chain (HC) are as follows:

LC CDR1: (SEQ ID NO: 14) TGTSSDVGGYNYVS LC CDR2: (SEQ ID NO: 15) DVSKRPSLC CDR3: (SEQ ID NO: 16) CSYAGSYTLV HC CDR1: (SEQ ID NO: 17) TYQMVHC CDR2: (SEQ ID NO: 18) VIYPSGGPTVYADSVKG HC CDR3: (SEQ ID NO: 19)GEDYYDSSGPGAFDI

Example 4 Exemplary MMP-9/2 Binding Antibody

M0237-D02. An exemplary MMP-9/2 antibody is M0237-D02. The amino acidsequences of variable regions of 539A-M0237-D02 sFAB are as follows:

539A-M0237-D02 (phage/SFAB) VL leader + VL (SEQ ID NO: 20)FYSHSAQDIQMTQSPATLSLSPGERATLSCRASQSISSFLAWYQQKPGQAPRLLIYDASYRATGIPARFSGSGSGTDFTLTISSLEPEDYAVYYCQQRGNWPITFGQGTRLEIKRTVAAPS  539A-M0237-D02 (phage/SFAB) VH leader + VH(SEQ ID NO: 21) MKKLLFAIPLVVPFVAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMWWVRQAPGKGLEWVSYIVPSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRAYGDYVGWNGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKS

Example 5 Exemplary MMP-14 Binding Antibodies

DX-2400. An exemplary MMP-14 antibody is DX-2400. The variable domainsequences for DX-2400 are:

VH: (SEQ ID NO: 22)FR1--------------------------- CDR1- FR2----------- CDR2------- DX-2400EVQLLESGGGLVQPGGSLRLSCAASGFTFS LYSMN WVRQAPGKGLEWVS SIYSSGGSTLYCDR2-- FR3----------------------------- CDR3-- FR4--------- DX-2400ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GRAFDI WGQGTMVTVSS CDR regionsare in bold.

VL: (SEQ ID NO: 23)FR1-------------------- CDR1------- FR2------------ CDR2--- DX-2400DIQMTQSPSSLSASVGDRVTITC RASQSVGTYLN WYQQKPGKAPKLLIY ATSNLRS GVPSFR3------------------------- CDR3------ FR4------- DX-2400RFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSIPRFT FGPGTKVDIK CDR regions are inbold.

DX-2410. Another exemplary MMP-14 antibody is DX-2410. The variabledomain sequences for DX-2410 are:

VH: (SEQ ID NO: 24)FR1--------------------------- CDR1- FR2----------- CDR2------- DX2410EVQLLESGGGLVQPGGSLRLSCAASGFTFS VYGMV WVRQAPGKGLEWVS VISSSGGSTWYCDR2-- FR3----------------------------- CDR3------- FR4-------- DX2410ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PFSRRYGVFDY WGQGTLVTVSS CRDregions are in bold.

VL: (SEQ ID NO: 25)FR1-------------------- CDR1------- FR2------------ CDR2--- DX2410DIQMTQSPSSLSASVGDRVTITC RASQGIRNFLA WYQQKPGKVPKLLIY GASALQSFR3----------------------------- CDR3----- FR4------- DX2410GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QKYNGVPLT FGGGTKVEIK CDR regions are inbold.

Example 6 Additional Exemplary MMP-9 Binding Antibodies

A protein containing the HC CDR sequences of 539A-M0240-B03 and thelight chain sequence shown below can be used in the methods describedherein. A protein containing the LC CDRs shown below and the HC CDRs of539A-M0240-B03, or a protein containing the LC variable region (light Vgene) shown below and the 539A-M0240-B03 HC CDRs can also be used in themethods described herein. The protein can include a constant regionsequence, such as the constant region (LC-lambda1) shown below.

Light V gene = VL2_2e; J gene = JL3 (SEQ ID NO: 26)    FR1-L                CDR1-L         FR2-L          CDR2-LQSALTQPRSVSGSPGQSVTISC TGTSSDVGGYNYVS WYQQHPGKAPKLMIY DVSKRPS GVPD      FR3-L                  CDR3-L     FR4-LRFSGSKSGNTASLTISGLQAEDEADYYC CSYAGSYTLV FGGGTKLTVL      ------------------- LC-lambda1 (SEQ ID NO: 27)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CDR regions are in bold.

The amino acid and nucleic acid sequences for another exemplary proteinthat can be used in the methods described herein are provided below. Aprotein containing the LC and HC CDRs shown below, or a proteincontaining the light chain and heavy chain variable regions (LV and HV,respectively) shown below can also be used in the methods describedherein.

Light Chain Light V gene = VL2_2e 2e.2.2/V1-3/DPL12 Light J gene = JL3

Heavy Chain Heavy V gene: VH3_3-23 DP-47/V3-23 Heavy J gene: JH3

Light VariableAntibody A-Light: Parental clone (sFab; IgG in pBh1 (f)) light variable Q  Y  E  L  T  Q  P  R  S  V  S  G  S  P  G  Q  S  V  T  I Antibody A:CAGTACGAATTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATC  Antibody A:

  Antibody A:

 P  D  R  F  S  G  S  K  S  G  N  T  A  S  L  T  I  S  G  L Antibody A:CCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTC  Antibody A:

 F  G  G  G  T  K  L  T  V  L (SEQ ID NO: 30) Antibody A:TTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO: 31) Heavy VariableAntibody A-Heavy: Parental clone (sFab; IgG in pBh1 (f)) Heavy variable E  V  Q  L  L  E  S  G  G  G  L  V  Q  P  G  G  S  L  R  L Antibody A:GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTT  Antibody A:

  Antibody A:

  Antibody A:

  Antibody A:

  Antibody A:

The amino acid and nucleic acid sequences for another exemplary proteinthat can be used in the methods described herein are provided below. Aprotein containing the LC and HC CDRs shown below, or a proteincontaining the light chain and heavy chain variable regions (LV and HV,respectively) shown below can also be used in the methods describedherein. A protein containing the light chain and heavy chain (designatedas LV+LC and HV+HC, respectively, below) sequences can also be used.

Light Chain Light V gene = VL2_2e 2e.2.2/V1-3/DPL12 Light J gene = JL3

Heavy Chain Heavy V gene: VH3_3-23 DP-47/V3-23 Heavy J gene: JH3

Light Variable Antibody B-Light: Germlined, codon optimized in GS vectorAntibody    CAGAGCGCCCTGACCCAGCCCAGAAGCGTGTCCGGCAGCCCAGGCCAGAGCGTGACCATCB:  Q  S  A  L  T  Q  P  R  S  V  S  G  S  P  G  Q  S  V  T  I Antibody B: 

Antibody B:

Antibody B: CCCGACAGGTTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACCATCTCCGGACTG P  D  R  F  S  G  S  K  S  G  N  T  A  S  L  T  I  S  G  L Antibody B:

Antibody B: TTCGGCGGAGGGACCAAGCTGACCGTGCTG (SEQ ID NO:  36) F  G  G  G  T  K  L  T  V  L  (SEQ ID NO:  37) Heavy VariableAntibody B-Heavy: Germlined, codon optimised in GS vector Antibody B:GAGGTGCAATTGCTGGAAAGCGGCGGAGGACTGGTGCAGCCAGGCGGCAGCCTGAGGCTG E  V  Q  L  L  E  S  G  G  G  L  V  Q  P  G  G  S  L  R  L Antibody B:

Antibody B:

Antibody B:

Antibody B:

Antibody B:

>Antibody B: LV + LC dnaCAGAGCGCCCTGACCCAGCCCAGAAGCGTGTCCGGCAGCCCAGGCCAGAGCGTGACCATCAGCTGCACCGGCACCAGCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAAGAGGCCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACCATCTCCGGACTGCAGGCCGAGGACGAGGCCGACTACTACTGCTGCAGCTACGCCGGCAGCTACACCCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTGCTGGGCCAGCCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACACTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACAACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGACAAAACCGTGGCCCCCACCCGAGTGTAGCTGATGA (SEQ ID NO: 40) >Antibody B: HV + HC dnaGAGGTGCAATTGCTGGAAAGCGGCGGAGGACTGGTGCAGCCAGGCGGCAGCCTGAGGCTGTCCTGCGCCGCCAGCGGCTTCACCTTCAGCACCTACCAGATGGTGTGGGTGCGCCAGGCCCCAGGCAAGGGCCTGGAATGGGTGTCCGTGATCTACCCCAGCGGCGGACCCACCGTGTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGAGGACTACTACGACAGCAGCGGCCCAGGCGCCTTCGACATCTGGGGCCAGGGCACAATGGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCGCTAGCACCTTCCTCCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCATACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGCCCTCCCTGCCCTGCCCCTGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTTAATTGGTATGTGGACGGCGTGGAGGTCCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTCAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTCCGGCAGCAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCTGGAAGTGA (SEQ ID NO: 41) >Antibody B: LV + LC aaQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSBNTASLTISGLQAEDEADYYCCSYAGSYTLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSss (SEQ ID NO: 42) >Antibody B: HV + HC aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYQMVWVRQAPGKGLEWVSVIYPSGGPTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARCEDYYDSSCPGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHRPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVXFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHPDWLNGKEYKCRVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKs (SEQ ID NO: 43)

REFERENCES

The contents of all cited references including literature references,issued patents, published or non-published patent applications citedthroughout this application are hereby expressly incorporated byreference in their entireties. In case of conflict, the presentapplication, including any definitions herein, will control.

EQUIVALENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-42. (canceled)
 43. A method for treating osteotropic cancer arisingfrom a metastatic cancer in a subject, the method comprisingadministering to the subject an effective amount of an antibody thatspecifically binds to a MMP-14 polypeptide comprising SEQ ID NO: 1,wherein the antibody inhibits the activity of MMP-14 and comprises aheavy chain immunoglobulin variable domain sequence and a light chainimmunoglobulin variable domain, wherein the heavy chain immunoglobulinvariable domain sequence comprises the heavy chain CDR1, CDR2 and CDR3of SEQ ID NO:22 and the light chain immunoglobulin variable domainsequence comprises the CDR1, CDR2 and CDR3 of SEQ ID NO: 23 to a subjectsuffering from an osteotropic cancer, thereby treating the osteotropiccancer in the subject.
 44. The method of claim 1, wherein the metastaticcancer is associated with metastatic breast cancer, metastatic lungcancer, metastatic renal cancer, multiple myeloma, and metastaticthyroid cancer.
 45. The method of claim 1, further comprisingadministering an additional therapeutic to the subject.
 46. The methodof claim 45, wherein the additional therapeutic is selected from thegroup consisting of an antitubulin/antimicrotubule agent, atopoisomerase I inhibitor, an antimetabolite, an alkylating agent, anapoptotic agent, and an anti-hormone.
 47. The method of claim 45,wherein the additional therapeutic is selected from the group consistingof: a bisphosphonate, a hormone-related compound, a RANKL antagonist, anα_(γ)β₃ antagonist, a Src inhibitor, a cathepsin K inhibitor andcalcitonin.
 48. A method for reducing the development of osteolyticlesions arising from a metastatic cancer in a subject, the methodcomprising administering to the subject an effective amount of anantibody that specifically binds to a MMP-14 polypeptide comprising SEQID NO: 1, wherein the antibody inhibits the activity of MMO-14 andcomprises a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence, wherein the heavychain immunoglobulin variable domain sequence comprises the heavy chainCDR1, CDR2 and CDR3 of SEQ ID NO:22 and the light chain immunoglobulinvariable domain sequence comprises the CDR1, CDR2 and CDR3 of SEQ IDNO:23, thereby reducing the development of said osteolytic lesions inthe subject.
 49. The method of claim 48, further comprisingadministering an additional therapeutic to the subject.
 50. The methodof claim 48, wherein the additional therapeutic is selected from thegroup consisting of an antitubulin/antimicrotubule agent, atopoisomerase I inhibitor, an antimetabolite, an alkylating agent, anapoptotic agent, and an anti-hormone.
 51. The method of claim 48,wherein the additional therapeutic is selected from the group consistingof: a bisphosphonate, a hormone-related compound, a RANKL antagonist, anα_(γ)β₃ antagonist, a Src inhibitor, a cathepsin K inhibitor andcalcitonin.
 52. The method of claim 1, wherein the antibody is anantibody fragment selected from the group consisting of a Fab fragment,a soluble Fab (sFab) fragment, an F(ab′)₂ fragment, an Fd fragment, andan Fv fragment.
 53. The method of claim 1, wherein the metastatic canceris associated with metastatic breast cancer, metastatic lung cancer,metastatic renal cancer, multiple myeloma, and metastatic thyroidcancer.
 54. The method of claim 1 or 48, wherein the metastatic canceris metastatic breast cancer.
 55. The method of claim 1 or 48, whereinthe antibody comprises the heavy chain variable region comprising SEQ IDNO:22.
 56. The method of claim 1 or 48, wherein the antibody comprisesthe light chain variable region comprising SEQ ID NO:23.
 57. The methodof claim 1 or 48, wherein the antibody comprises the heavy chainvariable region comprising SEQ ID NO:22 and the light chain variableregion comprising SEQ ID NO:23.
 58. The method of claim 1 or 48, whereinthe antibody is an IgG-1 antibody.
 59. The method of claim 1 or 48,wherein the antibody is a humanized antibody or a deimmunized antibody.60. The method of claim 1 or 48, wherein the antibody is physicallyassociated with a moiety that improves stabilization and/or retention inthe circulation.
 61. The method of claim 60, wherein the antibody isPEGylated.
 62. The method of claim 1 or 48, wherein the antibody is aprimate antibody or a chimeric antibody comprising a primate Fc domain.63. A kit comprising: a container comprising an antibody that binds SEQID NO: 1, the antibody comprising a heavy chain immunoglobulin variabledomain sequence and a light chain immunoglobulin variable domainsequence, wherein the heavy chain immunoglobulin variable domainsequence comprises the heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO:22and the light chain immunoglobulin variable domain sequence comprisesthe CDR1, CDR2 and CDR3 of SEQ ID NO:23 and instructions for use of saidantibody for the treatment of an osteotropic cancer arising from ametastatic cancer.